PBTK model-based analysis of CYP3A4 induction and the toxicokinetics of the pyrrolizidine alkaloid retrorsine in man.

Cytochrome P450 (CYP)3A4 induction by drugs and pesticides plays a critical role in the enhancement of pyrrolizidine alkaloid (PA) toxicity as it leads to increased formation of hepatotoxic dehydro-PA metabolites. Addressing the need for a quantitative analysis of this interaction, we developed a physiologically-based toxicokinetic (PBTK) model. Specifically, the model describes the impact of the well-characterized CYP3A4 inducer rifampicin on the kinetics of retrorsine, which is a prototypic PA and contaminant in herbal teas. Based on consumption data, the kinetics after daily intake of retrorsine were simulated with concomitant rifampicin treatment. Strongest impact on retrorsine kinetics (plasma AUC 24 and C max reduced to 67% and 74% compared to the rifampicin-free reference) was predicted directly after withdrawal of rifampicin. At this time point, the competitive inhibitory effect of rifampicin stopped, while CYP3A4 induction was still near its maximum. Due to the impacted metabolism kinetics, the cumulative formation of intestinal retrorsine CYP3A4 metabolites increased to 254% (from 10 to 25 nmol), while the cumulative formation of hepatic CYP3A4 metabolites was not affected (57 nmol). Return to baseline PA toxicokinetics was predicted 14 days after stop of a 14-day rifampicin treatment. In conclusion, the PBTK model showed to be a promising tool to assess the dynamic interplay of enzyme induction and toxification pathways.

Species difference in toxicokinetics and safety assessment of senecionine N-oxide in a UDP-glucuronosyltransferase 1A4 humanized mouse model.

Pyrrolizidine alkaloids (PAs) are naturally occurring hepatotoxins, and herbs containing PAs are of high concern. PAs are normally found in tertiary amines and N-oxide forms (PA N-oxides), yet the latter are less evaluated for their toxicokinetics. As a continuation of our investigation into the safety assessment of PA-containing herbal medicines, the toxicity and toxicokinetic characteristics of senecionine N-oxide (a representative toxic PA N-oxide) were investigated by using the UDP-glucuronosyltransferase 1A4 humanized mouse model (hUGT1A4 mouse model) and compared with those in wild-type mice simultaneously. Results show that the toxicity caused by senecionine N-oxide exposure was evidently decreased in hUGT1A4 mice as approved by pathology and biochemistry assays. In addition, a N-glucuronidation conjugate was exclusively found in hUGT1A4 mice but not in wild-type (WT) mice. In vitro studies proved that senecionine N-oxide initially reduced to the corresponding tertiary amine alkaloid (senecionine) and then underwent N-glucuronidation via human UGT1A4. The variation in toxicokinetic characteristics was also observed between hUGT1A4 mice and WT mice with a notably enhanced clearance of senecionine N-oxide and senecionine, and accordingly less formation of pyrrole-protein adducts in hUGT1A4 mice, which finally led to the detoxification of senecionine N-oxide exposure in hUGT1A4 mice. Our results provided the first in vivo toxicity data and toxicokinetic characteristics of senecionine N-oxide in a humanized animal model and revealed that human UGT1A4 plays an important role in the detoxification of senecionine N-oxide.

Toxicokinetic and bioavailability studies on retrorsine in mice, and ketoconazole-induced alteration in toxicokinetic properties.

Retrorsine (RTS) is a toxic retronecine-type pyrrolizidine alkaloid, which is widely distributed. The purpose of this study was to develop a high-performance liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for serum RTS determination in mice. Serum samples were deproteinated by acetonitrile, separated on a C18 -PFP column and delivered at 0.8 ml/min with an eluting system composed of water containing 0.1% (v/v) formic acid and acetonitrile containing 0.1% (v/v) formic acid as mobile phases. RTS and the internal standard S-hexylglutathione (H-GSH) were quantitatively monitored with precursor-to-product transitions of m/z 352.1 → 120.1 and m/z 392.2 → 246.3, respectively. The method showed excellent linearity over the concentration range 0.05-50 µg/ml, with correlation coefficient r2 = 0.9992. The extraction recovery was >86.34%, and the matrix effect was not significant. Inter- and intra-day precisions (RSD) were <4.99%. The validated LC-MS/MS method was successfully applied to study the toxicokinetic profiles of serum RTS in mice after intravenous, oral administration and co-treated with ketoconazole, which showed that RTS displayed a long half-life (~11.05 h) and good bioavailability (81.80%). Co-administration of ketoconazole (KTZ) increased the peak serum concentration and area under the concentration-time curve and decreased the clearance and mean residence time. Summing up, a new standardized method was established for quantitative determination of RTS in sera.

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.

LC-MS/MS method for determination of seneciphylline and its metabolite, seneciphylline N-oxide in rat plasma, and its application to a rat pharmacokinetic study.

A rapid and sensitive ultra-performance liquid chromatography-tandem electrospray ionization mass spectrometry (UPLC-ESI/MS) method was established and validated for simultaneous determination of seneciphylline and its main metabolite in rat plasma. The plasma sample was prepared by simple methanol-mediated precipitation. Chromatographic separation was achieved within 3 min by gradient elution using acetonitrile and water containing 0.1% formic acid as mobile phase on a Waters ACQUITY BEH C18 column (100 × 2.1 mm, i.d. 1.7 µm). Quantitation was conducted in a positive multiple reaction monitoring mode. The linearity of the method was over the range of 1-1,000 ng/mL, with the lower limit of quantification of 1 ng/mL. The intra- and inter-day precision and accuracy, extraction recovery, and matrix effect of analytes were within the acceptable limit. The analytes were stable during the process of sample collection, preparation, and analysis. The validated method was further applied to a pharmacokinetic study of seneciphylline in rats after oral and intravenous administration. The results revealed that seneciphylline was quickly absorbed into plasma (Tmax , 0.23-0.32 h) and reached the maximum concentration of 0.82-1.75 µg/mL after oral administration. Both seneciphylline and seneciphylline N-oxide were eliminated from plasma quickly. The low system exposure (oral bioavailability, 5.43-10.31%) was related to the extensive metabolism in the liver and microflora.

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.

Radioiodination and in vivo assessment of the potential of newly synthesized pyrrolizine-5-carboxamides derivative in tumor model.

Recently, pyrrolizine derivatives have been reported to possess numerous anticancer activities. In a previous study, (EZ)-6-((4-chlorobenzylidene)-amino)-7-cyano-N-(p-tolyl)-2,3-dihydro-1H-pyrrolizine carboxamide (EZPCA) compound was synthesized and the cytotoxic activity of EZPCA toward COX-2 enzyme (overexpressed in cancer cells) was reported. In order to assess the suitability of this compound as a promising pilot structure for in vivo applications, EZPCA was radiolabeled with radioiodine-131 (131I) and various factors affecting radiolabeling process were studied. Quality control studies of [131I]iodo-EZPCA were performed using paper chromatography and HPLC was used as a co-chromatographic technique for confirming the radiochemical yield. Biodistribution studies of [131I]iodo-EZPCA were undertaken in normal and tumor bearing mice. The radiochemical yield percentage of [131I]iodo-EZPCA was 94.20 ± 0.12%. The biodistribution results showed evident tumor uptake of [131I]iodo-EZPCA with promising target/non-target (T/NT) ratios. As a conclusion, these data suggest that [131I]iodo-EZPCA had high binding efficiency, high tumor uptake and sufficient stability to be used be used in diagnostic studies.

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.

The pyrrolizidine alkaloid senecionine induces CYP-dependent destruction of sinusoidal endothelial cells and cholestasis in mice.

Pyrrolizidine alkaloids (PAs) are widely occurring phytotoxins which can induce severe liver damage in humans and other mammalian species by mechanisms that are not fully understood. Therefore, we investigated the development of PA hepatotoxicity in vivo, using an acutely toxic dose of the PA senecionine in mice, in combination with intravital two-photon microscopy, histology, clinical chemistry, and in vitro experiments with primary mouse hepatocytes and liver sinusoidal endothelial cells (LSECs). We observed pericentral LSEC necrosis together with elevated sinusoidal marker proteins in the serum of senecionine-treated mice and increased sinusoidal platelet aggregation in the damaged tissue regions. In vitro experiments showed no cytotoxicity to freshly isolated LSECs up to 500 µM senecionine. However, metabolic activation of senecionine by preincubation with primary mouse hepatocytes increased the cytotoxicity to cultivated LSECs with an EC50 of approximately 22 µM. The cytochrome P450 (CYP)-dependency of senecionine bioactivation was confirmed in CYP reductase-deficient mice where no PA-induced hepatotoxicity was observed. Therefore, toxic metabolites of senecionine are generated by hepatic CYPs, and may be partially released from hepatocytes leading to destruction of LSECs in the pericentral region of the liver lobules. Analysis of hepatic bile salt transport by intravital two-photon imaging revealed a delayed uptake of a fluorescent bile salt analogue from the hepatic sinusoids into hepatocytes and delayed elimination. This was accompanied by transcriptional deregulation of hepatic bile salt transporters like Abcb11 or Abcc1. In conclusion, senecionine destroys LSECs although the toxic metabolite is formed in a CYP-dependent manner in the adjacent pericentral hepatocytes.

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.

Integrating physiologically based kinetic (PBK) and Monte Carlo modelling to predict inter-individual and inter-ethnic variation in bioactivation and liver toxicity of lasiocarpine.

The aim of the present study was to predict the effect of inter-individual and inter-ethnic human kinetic variation on the sensitivity towards acute liver toxicity of lasiocarpine in the Chinese and the Caucasian population, and to derive chemical specific adjustment factors (CSAFs) by integrating variation in the in vitro kinetic constants Vmax and Km, physiologically based kinetic (PBK) modelling and Monte Carlo simulation. CSAFs were derived covering the 90th and 99th percentile of the population distribution of pyrrole glutathione adduct (7-GS-DHP) formation, reflecting bioactivation. The results revealed that in the Chinese population, as compared to the Caucasian population, the predicted 7-GS-DHP formation at the geometric mean, the 90th and the 99th percentile were 2.1-, 3.3- and 4.3-fold lower respectively. The CSAFs obtained using the 99th percentile values were 8.3, 17.0 and 19.5 in the Chinese, the Caucasian population and the two populations combined, respectively, while the CSAFs were generally 3.0-fold lower at the 90th percentile. These results indicate that when considering the formation of 7-GS-DHP the Caucasian population may be more sensitive towards acute liver toxicity of lasiocarpine, and further point out that the default safety factor of 3.16 for inter-individual human kinetic differences may not be sufficiently protective. Altogether, the results obtained demonstrate that integrating PBK modelling with Monte Carlo simulations using human in vitro data is a powerful strategy to quantify inter-individual variations in kinetics, and can be used to refine the human risk assessment of pyrrolizidine alkaloids.

Intestinal and hepatic biotransformation of pyrrolizidine alkaloid N-oxides to toxic pyrrolizidine alkaloids.

Pyrrolizidine alkaloids (PAs) are among the most significant groups of phytotoxins present in more than 6000 plants in the world. Hepatotoxic retronecine-type PAs and their corresponding N-oxides usually co-exist in plants. Although PA-induced hepatotoxicity is known for a long time and has been extensively studied, the toxicity of PA N-oxide is rarely investigated. Recently, we reported PA N-oxide-induced hepatotoxicity in humans and rodents and also suggested the association of such toxicity with metabolic conversion of PA N-oxides to the corresponding toxic PAs. However, the detailed biochemical mechanism of PA N-oxide-induced hepatotoxicity is largely unknown. The present study investigated biotransformation of four representative cyclic retronecine-type PA N-oxides to their corresponding PAs in both gastrointestinal tract and liver. The results demonstrated that biotransformation of PA N-oxides to PAs was mediated by both intestinal microbiota and hepatic cytochrome P450 monooxygenases (CYPs), in particular CYP1A2 and CYP2D6. Subsequently, the formed PAs were metabolically activated predominantly by hepatic CYPs to form reactive metabolites exerting hepatotoxicity. Our findings delineated, for the first time, that the metabolism-mediated mechanism of PA N-oxide intoxication involved metabolic reduction of PA N-oxides to their corresponding PAs in both intestine and liver followed by oxidative bioactivation of the resultant PAs in the liver to generate reactive metabolites which interact with cellular proteins leading to hepatotoxicity. In addition, our results raised a public concern and also encouraged further investigations on potentially remarkable variations in PA N-oxide-induced hepatotoxicity caused by significantly altered intestinal microbiota due to individual differences in diets, life styles, and medications.

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

SCOPE: 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. METHODS AND RESULTS: 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. CONCLUSION: 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.

Pyrrolizidine alkaloid-induced alterations of prostanoid synthesis in human endothelial cells.

Pyrrolizidine alkaloids (PA) are a group of secondary plant metabolites belonging to the most widely distributed natural toxins. PA intoxication of humans leads to severe liver damage, such as hepatomegaly, hepatic necrosis, fibrosis and cirrhosis. An acute consequence observed after ingestion of high amounts of PA is veno-occlusive disease (VOD) where the hepatic sinusoidal endothelial cells are affected. However, the mechanisms leading to VOD after PA intoxication remain predominantly unknown. Thus, we investigated PA-induced molecular effects on human umbilical vein endothelial cells (HUVEC). We compared the effects of PA with the effects of PA metabolites obtained by in vitro metabolism using liver homogenate (S9 fraction). In vitro-metabolized lasiocarpine and senecionine resulted in significant cytotoxic effects in HUVEC starting at 300 µM. Initial molecular effect screening using a PCR array with genes associated with endothelial cell biology showed PA-induced upregulation of the Fas receptor, which is involved in extrinsic apoptosis, and regulation of a number of interleukins, as well as of different enzymes relevant for prostanoid synthesis. Modulation of prostanoid synthesis was subsequently studied at the mRNA and protein levels and verified by increased release of prostaglandin I2 as the main prostanoid of endothelial cells. All effects occurred only with in vitro-metabolically activated PA lasiocarpine and senecionine. By contrast, no effect was observed for the PA echimidine, heliotrine, lasiocarpine, senecionine, senkirkine and platyphylline in the absence of an external metabolizing system up to the highest tested concentration of 500 µM. Overall, our results confirm the metabolism-dependent toxification of PA and elucidate the involved pathways. These include induction of inflammatory cytokines and deregulation of the prostanoid synthesis pathway in endothelial cells, linking for the first time PA-dependent changes in prostanoid release to distinct alterations at the mRNA and protein levels of enzymes of prostanoid synthesis.

Relative potency of fifteen pyrrolizidine alkaloids to induce DNA damage as measured by micronucleus induction in HepaRG human liver cells.

Plant-based 1,2-unsaturated Pyrrolizidine Alkaloids (PAs) can be found as contaminants in foods like teas, herbs and honey. PAs are responsible for liver genotoxicity/carcinogenicity following metabolic activation, making them a relevant concern for safety assessment. Current regulatory risk assessments take a precautionary approach and assume all PAs are as potent as the known most potent representatives: lasiocarpine and riddelliine. Our study investigated whether genotoxicity potency differed as a consequence of structural differences, assessing micronuclei in vitro in HepaRG cells which express metabolising enzymes at levels similar to primary human hepatocytes. Benchmark Dose (BMD) analysis was used to calculate the critical effect dose for 15 PAs representing 6 structural classes. When BMD confidence intervals were used to rank PAs, lasiocarpine was the most potent PA and plotted distinctly from all other PAs examined. PA-N-oxides were least potent, notably less potent than their corresponding parent PA's. The observed genotoxic potency compared favorably with existing in vitro data when metabolic competency was considered. Although further consideration of biokinetics will be needed to develop a robust understanding of relative potencies for a realistic risk assessment of PA mixtures, these data facilitate understanding of their genotoxic potencies and affirm that not all PAs are created equal.

Use of physiologically based kinetic modelling-facilitated reverse dosimetry to convert in vitro cytotoxicity data to predicted in vivo liver toxicity of lasiocarpine and riddelliine in rat.

Lasiocarpine and riddelliine are pyrrolizidine alkaloids (PAs) present in food and able to cause liver toxicity. The aim of this study was to investigate whether physiologically based kinetic (PBK) modelling-facilitated reverse dosimetry can adequately translate in vitro concentration-response curves for toxicity of lasiocarpine and riddelliine to in vivo liver toxicity data for the rat. To this purpose, PBK models were developed for lasiocarpine and riddelliine, and predicted blood concentrations were compared to available literature data to evaluate the models. Concentration-response curves obtained from in vitro cytotoxicity assays in primary rat hepatocytes were converted to in vivo dose-response curves from which points of departure (PODs) were derived and that were compared to available literature data on in vivo liver toxicity. The results showed that the predicted PODs fall well within the range of PODs derived from available in vivo toxicity data. To conclude, this study shows the proof-of-principle for a method to predict in vivo liver toxicity for PAs by an alternative testing strategy integrating in vitro cytotoxicity assays with in silico PBK modelling-facilitated reverse dosimetry. The approach may facilitate prediction of acute liver toxicity for the large number of PAs for which in vivo toxicity data are lacking.

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.

In vitro biotransformation of pyrrolizidine alkaloids in different species. Part I: Microsomal degradation.

Pyrrolizidine alkaloids (PA) are secondary metabolites of certain flowering plants. The ingestion of PAs may result in acute and chronic effects in man and livestock with hepatotoxicity, mutagenicity, and carcinogenicity being identified as predominant effects. Several hundred PAs sharing the diol pyrrolizidine as a core structure are formed by plants. Although many congeners may cause adverse effects, differences in the toxic potency have been detected in animal tests. It is generally accepted that PAs themselves are biologically and toxicologically inactive and require metabolic activation. Consequently, a strong relationship between activating metabolism and toxicity can be expected. Concerning PA susceptibility, marked differences between species were reported with a comparatively high susceptibility in horses, while goat and sheep seem to be almost resistant. Therefore, we investigated the in vitro degradation rate of four frequently occurring PAs by liver enzymes present in S9 fractions from human, pig, cow, horse, rat, rabbit, goat, and sheep liver. Unexpectedly, almost no metabolic degradation of any PA was observed for susceptible species such as human, pig, horse, or cow. If the formation of toxic metabolites represents a crucial bioactivation step, the found inverse conversion rates of PAs compared to the known susceptibility require further investigation.

Intermedin Ameliorates Atherosclerosis by Increasing Cholesterol Efflux Through the cAMP-PKA Pathway in Macrophage RAW264.7 Cell Line.

BACKGROUND The aim of this study was to explore the role of intermedin and its mechanism in cholesterol efflux of macrophage THP-1 and RAW264.7 cell lines in atherosclerosis (AS). MATERIAL AND METHODS ApoE-/- mice were fed with a high-fat diet, and the concentrations of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were measured. The lipidoses of the aortic sinus were analyzed by hematoxylin and eosin staining, and the cAMP level was detected by enzyme-linked immunosorbent assay (ELISA). The expressions of ATP-binding cassette transporter (ABCA1) were tested by real-time PCR and Western blot analysis. RESULTS IMD decreased serum TC and LDL-C, and increased serum HDL-C level in apoE-/- mice and attenuated AS plaque areas. In vitro, IMD increased intracellular cAMP concentration in a dose-dependent manner in THP-1 and RAW264.7 cell lines, which enhanced the expression of ABCA1 and increased cholesterol efflux rate. However, this effect was inhibited by PKAI in the RAW 264.7 cell line but not in the THP-1 cell line. CONCLUSIONS IMD can ameliorate the development of AS in ApoE-/- mice and regulate cholesterol balance in the RAW264.7 cell line through the cAMP-PKA pathway.

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.