The toxicokinetic and metabolism of structurally diverse pyrrolizidine alkaloids in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Pyrrolizidine alkaloids (PAs) are a group of phytotoxins present in about 3% of flowering plants worldwide. Ingestion of PA-containing herbal products may lead to hepatotoxicity. Notably, the toxicokinetic (TK) behaviors, especially pyrrole-protein adducts (PPAs) having the same structure but generated from metabolic activation of different PAs, significantly affect the toxicity of structurally diverse PAs, therefore studying them in their pure form is preferable to extracts to stratify toxic potency of different PAs co-existing in herbal extracts. However, previous studies mainly focus on the establishment of TK profiles of the intact PAs, revealing less or no kinetic information on the main PA metabolites (PA N-oxides) and PPAs which mediate PA-induced hepatotoxicity. In this study, PPA was measured as the biomarker of PA exposure and PA-induced toxicity. AIM OF STUDY: This study aims to investigate the TK difference between structurally diverse PAs of retronecine-type PAs: retrorsine (RTS) and monocrotaline (MCT), and otonecine-type PA: clivorine (CLI), and their toxicity-related metabolite PPAs and PA N-oxides, the main metabolite of retronecine-type PAs, for the establishment of a more accurate risk assessment of PAs exposure. MATERIALS AND METHODS: The TK studies were conducted using rats through intravenous (i.v.) or oral (p.o.) administration of PAs at 20 mg/kg. The main TK parameters of PAs and PA N-oxides were determined from plasma concentration-time profiles, and the kinetic profiles of PPAs were assessed from both plasma and erythrocyte concentration-time profiles. RESULTS: MCT demonstrated the slowest but the highest extent of absorption among the three PAs, while RTS demonstrated a similar absorption rate with a lower extent than CLI. For elimination, MCT demonstrated a similar elimination rate as RTS but the lowest extent of elimination among the three PAs, and CLI exhibited significantly faster elimination than MCT and RTS. Moreover, the formation of PA N-oxide, which only occurs in retronecine-type PAs, was remarkably less in MCT-treated rats compared to RTS-treated ones. Of note, the retronecine-type RTS and MCT induced more PPAs via p.o. than i.v. administration route, whereas the otonecine-type CLI showed the opposite trend. CONCLUSION: Dramatic TK differences, including not only PAs but also PA N-oxides and the derived protein adduct PPAs, were found among structurally diverse PAs in rats, laying the basis for varied hepatotoxic potencies induced by different PA-containing herbal products. Notably, our findings for the first time uncovered that oral administration of retronecine-type PAs might cause severer toxicity compared with the intravenous route, which warrants further in-depth exploration.

Formation of DHP-DNA Adducts from Rat Liver Microsomal Metabolism of 1,2-Unsaturated Pyrrolizidine Alkaloid-Containing Plant Extracts and Dietary Supplements.

1,2-Unsaturated pyrrolizidine alkaloids (PAs) are carcinogenic phytochemicals. We previously determined that carcinogenic PAs and PA N-oxides commonly form a set of four (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-DNA adducts, namely, DHP-dG-3, DHP-dG-4, DHP-dA-3, and DHP-dA-4. This set of DHP-DNA adducts has been implicated as a potential biomarker of PA-induced liver tumor initiation from metabolism of individual carcinogenic PAs. To date, it is not known whether this generality occurs from metabolism of PA-containing plant extracts. In this study, we investigate the rat liver microsomal metabolism of nine PA-containing plant extracts and two PA-containing dietary supplements in the presence of calf thymus DNA. The presence of carcinogenic PAs and PA N-oxides in plant extracts was first confirmed by LC-MS/MS analysis with selected reaction monitoring mode. Upon rat liver microsomal metabolism of these PA-containing plant extracts and dietary supplements, the formation of this set of DHP-DNA adducts was confirmed. Thus, these results indicate that metabolism of PA-containing plant extracts and dietary supplements can generate DHP-dG-3, DHP-dG-4, DHP-dA-3, and DHP-dA-4 adducts, thereby potentially initiating liver tumor formation.

Nrf2-mediated liver protection by 18ß-glycyrrhetinic acid against pyrrolizidine alkaloid-induced toxicity through PI3K/Akt/GSK3ß pathway.

BACKGROUND: Misusage of pyrrolizidine alkaloid (PA)-containing plants or unaware intake of PA-contaminated foodstuffs causes thousands of PA poisoning cases in humans. PA intoxication is accompanied by oxidative stress and subsequent extensive hepatocellular damage. Our previous study has demonstrated that 18ß-glycyrrhetinic acid (GA), a bioactive constituent of liquorice, prevented PA-induced hepatotoxicity in rats, however the underlying mechanisms remain unclear. OBJECTIVE: This study aims to explore the mechanisms underlying the hepato-protective effect of GA in combating retrorsine (RTS, a representative toxic PA)-induced liver injury. METHODS: Histological and biochemical assessments were employed to evaluate the protective effect of GA on RTS-induced hepatotoxicity in rats. Sulforhodamine B assay, real-time PCR, western blotting, and immunostaining were used to explore the underlying mechanisms in human hepatocytes and rats. RESULTS: Our findings demonstrated that GA alleviated RTS-induced elevation of serum ALT and bilirubin levels, as well as hepatocytes necrosis and sinusoidal endothelial cells (SECs) damage in rats. GA also enhanced the activities and expressions of several antioxidant enzymes through upregulating nuclear factor-erythroid 2-related factor2 (Nrf2). Moreover, inhibition of Nrf2 blocked the hepatoprotective effect of GA against RTS intoxication. Mechanistically, GA increased the phosphorylation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and enhanced glycogen synthase kinase 3 beta (GSK3ß) inhibitory phosphorylation at serine 9, thus promoting the nuclear accumulation of Nrf2 and activating its downstream targets. CONCLUSION: This study for the first time demonstrated that GA exerted protective effects against RTS-induced liver injury by potentiating the Nrf2-mediated antioxidant system through PI3K/Akt/GSK3ß pathway. The findings indicated that GA may serve as a potential candidate drug for the treatment of PA intoxication.

Liquorice Extract and 18ß-Glycyrrhetinic Acid Protect Against Experimental Pyrrolizidine Alkaloid-Induced Hepatotoxicity in Rats Through Inhibiting Cytochrome P450-Mediated Metabolic Activation.

Misuse of pyrrolizidine alkaloid (PA)-containing plants or consumption of PA-contaminated foodstuffs causes numerous poisoning cases in humans yearly, while effective therapeutic strategies are still limited. PA-induced liver injury was initiated by cytochrome P450 (CYP)-mediated metabolic activation and subsequent formation of adducts with cellular proteins. Liquorice, a hepato-protective herbal medicine, is commonly used concurrently with PA-containing herbs in many compound traditional Chinese medicine formulas, and no PA-poisoning cases have been reported with this combination. The present study aimed to investigate hepato-protective effects of liquorice aqueous extract (EX) and 18ß-glycyrrhetinic acid (GA, the primary bioactive constituent of liquorice) against PA-induced hepatotoxicity and the underlying mechanism. Histopathological and biochemical analysis demonstrated that both single- and multiple-treatment of EX (500 mg/kg) or GA (50 mg/kg) significantly attenuated liver damage caused by retrorsine (RTS, a representative hepatotoxic PA). The formation of pyrrole-protein adducts was significantly reduced by single- (30.3% reduction in liver; 50.8% reduction in plasma) and multiple- (32.5% reduction in liver; 56.5% reduction in plasma) treatment of GA in rats. Single- and multiple-treatment of EX also decreased the formation of pyrrole-protein adducts, with 30.2 and 31.1% reduction in rat liver and 51.8 and 53.1% reduction in rat plasma, respectively. In addition, in vitro metabolism assay with rat liver microsomes demonstrated that GA reduced the formation of metabolic activation-derived pyrrole-glutathione conjugate in a dose-dependent manner with the estimated IC50 value of 5.07 µM. Further mechanism study showed that GA inhibited activities of CYPs, especially CYP3A1, the major CYP isoform responsible for the metabolic activation of RTS in rats. Enzymatic kinetic study revealed a competitive inhibition of rat CYP3A1 by GA. In conclusion, our findings demonstrated that both EX and GA exhibited significant hepato-protective effects against RTS-induced hepatotoxicity, mainly through the competitive inhibition of CYP-mediated metabolic activation of RTS.

Characterization of liver injury induced by a pyrrolizidine alkaloid in rats.

BACKGROUND: Pyrrolizidine alkaloids (PAs) are common phytotoxins. PA intoxication is reported to cause severe acute liver damage, typically known as hepatic sinusoidal obstruction syndrome (HSOS), but it remains obscure whether the acute liver damage may progress into chronic liver disease characterized by hepatic fibrosis. PURPOSE: This study aims to characterize the biochemical markers of liver injury and histological features of regressive and progressive liver fibrosis, and to examine changes in hepatic gene expression that may underpin mechanisms of fibrogenesis in rats induced by retrorsine (RTS), a representative toxic PA. STUDY DESIGN/METHODS: Rats were gavaged with RTS via two dosing regimens, i.e. a single dose of 40 mg/kg (Group 1) and two doses of 40 mg/kg and 20 mg/kg on day 0 and day 7 (Group 2), respectively. Rats receiving one (Group 3) or two (Group 4) doses of vehicle served as negative controls. The animals were followed for up to 16 weeks by serum biochemical analyses and histological examination, and gene expression assays of liver tissues. RESULTS: Acute liver injury on day 2 manifested as HSOS, characterized by sinusoidal dilation, endothelial cell damage, and elevated serum alanine aminotransferase activity and bilirubin levels. In Group 1, mild liver fibrosis developed at sinusoids and perisinusoidal space surrounding the central veins at week 1 and 2, and thereafter, all liver injury resolved gradually. In Group 2, liver fibrosis progressed within the 16-week observation period. No apparent liver injury was observed in Groups 3 and 4. Compared with negative control groups, RTS induced myofibroblastic activation, TGF-ß1 signaling, and changes in expression of matrix metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1). These dynamic changes differed in Groups 1 and 2, corresponding with the regression and progression of liver fibrosis, respectively, in these groups. CONCLUSION: This study has provided in-vivo proof of concept that "one hit" and "two hits" of RTS lead to acute resolving liver injury and chronic progressive liver fibrosis, respectively. These animal models may serve as powerful tools for studying RTS toxicology and related preventive and therapeutic strategies and as positive controls for studying other PA- and non-PA-induced liver injury.

Excessive Intake of Longan Arillus Alters gut Homeostasis and Aggravates Colitis in Mice.

Background: Longan is the fruit of Dimocarpus longan Lour. and the longan arillus has long been used in traditional Chinese medicine possessing various health benefits. However, the excessive intake of longan is found in daily life to cause "shanghuo" syndrome. "Shanghuo" has been linked to increased disease susceptibility. The present study thus aimed to investigate the toxicological outcomes after excessive longan treatment. Methods: Longan extract at a normal dosage of 4 g/kg and two excess dosages of 8 and 16 g/kg was orally administered to normal C57BL/6J mice for two weeks or to C57BL/6J mice with DSS-induced colitis. Mouse gut microbiome were analyzed by 16S rRNA sequencing. Short chain fatty acid (SCFA) contents in colonic contents were measured by GC-MS. Colon tissue was used for histopathological observation after H and E staining, detection of protein expression by western blot, analysis of gene expression by qPCR, and detection of apoptotic cells by TUNEL assay. ELISA was used for biochemical analysis in serum. Results: In normal mice, repeated longan intake at excess doses, but not the normal dose, increased infiltration of inflammatory cells, elevated serum levels of TNF-α and IL-6 and reduced production of SCFAs. In DSS-induced colitic mice, longan intake at 4 g/kg did not promote colitis in mice, while excessive longan (8 or 16 g/kg) aggravated colitis in mice, showing increased inflammation, more serious histological abnormalities, increased gut permeability, and increased epithelia injury when compared to DSS alone. Excessive longan induced a significant reduction of microbial diversity in colitic mice, accompanied with aggravated alterations of DSS-associated bacteria including the increase of Proteobacteria phylum and genera of Bacteroides, Akkermansia, Turicibacter and Escherchia-Shigella, and the decrease of norank_f__Muribaculaceae. The changed microbial compositions were accompanied with decreased SCFAs when longan was supplemented with DSS. The aggravated colon injury by excessive intake of longan in colitic mice was tightly correlated with the altered microbial communities and decreased SCFAs production. Conclusion: Excessive longan intake disturbs gut homeostasis and aggravates colitis via promoting inflammation and altering gut microbe compositions and associated metabolism in mice. Our findings warrant rational longan arillus consumption as a dietary supplement or herbal medicine.

The dual roles of ginsenosides in improving the anti-tumor efficiency of cyclophosphamide in mammary carcinoma mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Cyclophosphamide (CTX) is a first line chemotherapeutic agent, but often limited for its unstable therapeutic effect and serious side effects. Ginsenosides could facilitate the anti-tumor efficiency of CTX, including benefiting therapeutic effect and decreasing side effects. AIM OF THE STUDY: To investigate the potential mechanism of ginsenosides on benefiting the anti-tumor efficiency of CTX. MATERIALS AND METHODS: Mammary carcinoma mice were applied to investigate the anti-tumor efficiency and potential mechanism of combinational treatment of ginsenosides and CTX. Therapeutic effect was evaluated based on survival rate, tumor burden, tumor growth inhibition rate, and apoptosis and histological changes of tumor tissues. Anti-tumor immunity was studied by measuring serum level of anti-tumor cytokines. Gut mucositis, one of lethal side effects of CTX, was evaluated by diarrhea degree, gut permeability and tight junction proteins expressions. Gut microbial diversity was analyzed by 16S rRNA gene sequencing, and fecal transplant and antibiotics sterilized animals were performed to evaluate the therapeutic effect of gut microbiota on tumor suppression. RESULTS: Ginsenosides facilitated the therapeutic effect of CTX in mice, which manifested as prolonged survival rate, decreased tumor burden, as well as enhanced tumor growth inhibition rate and apoptosis. The favoring effect was related to elevation of anti-tumor immunity which manifested as the increased anti-tumor cytokines (INF-γ, IL-17, IL-2 and IL-6). Further studies indicated the elevation was ascribed to ginsenosides promoted reproduction of gut probiotics including Akkermansia, Bifidobacterium and Lactobacillus. Moreover, co-administration of ginsenosides in mice alleviated CTX-induced gut mucositis, including lower gut permeability, less diarrhea, less epithelium damage and higher tight junction proteins. Further researches suggested the alleviation was related to ginsenosides activated Nrf2 and inhibited NFκB pathways. CONCLUSION: Ginsenosides show dual roles to facilitate the anti-tumor efficiency of CTX, namely promote the anti-tumor immunity through maintaining gut microflora and ameliorate gut mucositis by modulating Nrf2 and NFκB pathways.

Pulmonary toxicity is a common phenomenon of toxic pyrrolizidine alkaloids.

The hepatotoxic pyrrolizidine alkaloids (PAs) are metabolically activated in the liver to form reactive dehydro-PAs, which generate pyrrole-protein adducts leading to hepatotoxicity. Monocrotaline, but not other PAs, is also pneumotoxic, supposedly due to the migration of the liver-generated corresponding dehydro-PA into the lung to form pyrrole-protein adducts to induce pneumotoxicity. The present study investigated whether other PAs are also pneumotoxic. Metabolic activation of four representative hepatotoxic PAs, monocrotaline, retrorsine, riddelliine and clivorine, was investigated using rat liver or lung S9 incubation. All PAs produced pyrrole-protein adducts significantly in rat liver S9 but negligible in lung S9 fraction, revealing that liver is the key organ responsible for metabolic activation generating dehydro-PAs. Furthermore, these four PAs and another two PAs present in the alkaloid extract of Gynura segetum, a widely used PA-producing herb responsible for human PA poisonings in China, were orally administered to rats using the same hepatotoxic dose of 0.2 mmol/kg. All six PAs induced pneumotoxicity in rats within 48 h. The results demonstrated that pneumotoxicity could be a common phenomenon of PAs and the liver-derived dehydro-PAs might move to the lung and form pyrrole-protein adducts, leading to pulmonary toxicity.

Pharmacokinetic profiles of falcarindiol and oplopandiol in rats after oral administration of polyynes extract of Oplopanax elatus.

Polyynes, such as facarindiol (FAD) and oplopandiol (OPD), are responsible for anticancer activities of Oplopanax elatus (O. elatus). A novel approach to pharmacokinetics determination of the two natural polyynes in rats was developed and validated using a liquid chromatography-electrospray ionization-mass spectrometry (LC-MS) method. Biosamples were prepared by liquid-liquid extraction using ethyl acetate/n-hexane (V : V = 9 : 1) and the analytes were eluted on an Agilent ZORBAX Eclipse Plus C18 threaded column (4.6 mm × 50 mm, 1.8 µm) with the mobile phase of acetonitrile-0.1% aqueous formic acid at a flow-rate of 0.5 mL·min(-1) within a total run time of 11 min. All analytes were simultaneously monitored in a single-quadrupole mass spectrometer in the selected ion monitoring (SIM) mode using electrospray source in positive mode. The method was demonstrated to be rapid, sensitive, and reliable, and it was successfully applied to the pharmacokinetic studies of the two polyynes in rat plasma after oral administration of polyynes extract of O. elatus.

Hepatotoxic assessment of Polygoni Multiflori Radix extract and toxicokinetic study of stilbene glucoside and anthraquinones in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Polygoni Multiflori Radix (PMR) has been traditionally used as a tonic and an anti-aging remedy for centuries; however, hepatic lesions linked to PMR have been frequently reported. AIM OF THE STUDY: This work attempted to investigate the hepatotoxic potential of PMR extract and the toxicokinetics of stilbene glucoside and anthraquinones in PMR extract following repeated administration. MATERIALS AND METHODS: Histopathological and biochemical tests were performed to assess the hepatotoxicity of PMR extract. A rapid and sensitive liquid chromatography-mass spectrometry (LC-MS) assay was developed for toxicokinetic analysis of the main constituents of PMR extract, including 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside (TSG), emodin-8-O-ß-D-glucoside and emodin. RESULTS: The histopathological and biochemical tests indicated that repeated administration of high-dose PMR extract (20 g/kg) for 3 weeks could cause hepatic lesions, while the low-dose treatment (1 g/kg) was safe. Necrosis and steatosis of hepatic cells, inflammatory cell infiltration and mild fibrosis were the main toxicity symptoms caused by high-dose PMR extract in rat liver. The aspartate aminotransferase (AST) levels increased by approximately 17%, from 110.80±0.84 to 129.75±10.83 IU/L, in the high-dose group compared with the control group. The proposed LC-MS method was proven to be suitable for the simultaneous quantification of these three constituents by affording desirable linearity (r(2)>0.998) and satisfactory precision (error less than 10%). The toxicokinetic study showed that emodin could not be detected in the low-dose group, but the AUC and Cmax of emodin displayed a gradual increase with repeated treatments in the high-dose group. The toxicokinetics of TSG in the low- and high-dose groups exhibited similar trends after repeated administration. CONCLUSIONS: Consideration needs to be given to the rational application of PMR in the clinic to balance its benefits and risks. The increased emodin exposure in vivo provided a putative explanation for the observed hepatic lesions induced by PMR extract, although further studies to confirm the potentially causal link between emodin exposure and hepatic lesions are still necessary.

Effects of American ginseng on pharmacokinetics of 5-fluorouracil in rats.

The pharmacokinetics of 5-fluorouracil (5-FU) in combination with or without American ginseng (seven-consecutive days oral dose) in rats were evaluated using liquid chromatography-electrospray ionization-mass spectrometry (LC-MS). Chromatographic separation was performed on a reverse LC column within a total run time of 6.5 min, which allowed for a relatively quick analysis. The limit of quantification for 5-FU was 15 ng/mL and this method was linear over 15-50,000 ng/mL. This method supported stabilizing determination of the plasma concentration of 5-FU over a period of 24 h. Precision both interday and intraday (coefficient of variation) was within 14% and accuracy (relative error) ranged from -5 to 14%. In view of the observed pharmacokinetic parameters, including maximum concentration, time to maximum concentration, area under the concentration-time curve (AUC), mean residence time, elimination half-life and clearance, our results showed no significant differences in all of the pharmacokinetic parameters between the ginseng co-treated group and 5-FU alone group. Some increase in AUC was observed in 5-FU plus ginseng group; however, the difference did not reach statistical significance compared with 5-FU alone. It appeared that American ginseng administration did not significantly alter the kinetics of 5-FU. More studies are still needed to confirm our results.

Metabonomic analysis of the therapeutic effect of Potentilla discolor in the treatment of type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM) is increased worldwide in parallel with the obesity epidemic. Potentilla discolor is one of the most important crude materials in Traditional Chinese medicine (TCM) for therapy of hyperglycemia and hyperlipidemia. In this work, a plasma metabonomic approach based on the combination of UPLC-Q-TOF with multivariate data analysis was applied to investigate the therapeutic effects of the extract of P. discolor (EPD) and corosolic acid (CA), the main bioactive compounds of P. discolor. Male C57BL/6 mice were fed with high-fat diet (HFD-fed group) for 8 weeks and then treated with EPD (EPD-treated group) or CA (CA-treated group) for another 8 weeks. After the experimental period, samples of plasma were collected and analyzed by ultra-performance liquid chromatography/quadrupole time of flight mass spectrometry (UPLC-Q-TOF). The principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) models were built to find biomarkers of T2DM and investigate the therapeutic effects of EPD and CA. 26 metabolites, which are distributed in several metabolic pathways, were identified as potential biomarkers of T2DM. It was found that EPD and CA could reverse the pathological process of T2DM through regulating the disturbed pathway of metabolism. The metabonomic results are beneficial not only for the evaluation of the therapeutic effect of TCM but also for the elucidation of the underlying molecular mechanism.

Stilbene glucoside inhibits the glucuronidation of emodin in rats through the down-regulation of UDP-glucuronosyltransferases 1A8: application to a drug-drug interaction study in Radix Polygoni Multiflori.

ETHNOPHARMACOLOGICAL RELEVANCE: The integrated effects of herbal medicines were the outcome of all of the inherent components. Currently, few studies have focused on the multicomponent interactions in an herbal medicine to elucidate its pharmacological and/or toxicological effects. In this study, an attempt was made to investigate the interaction between stilbene glucosides and the anthraquinones contained in Radix Polygoni Multiflori (RPM) and to explore the interaction's mechanism from the perspective of UDP-glucuronosyltransferase (UGT) regulation. MATERIALS AND METHODS: The extract of RPM was separated into a stilbene glucoside fraction and a emodin fraction. A rapid high-performance liquid chromatography-mass spectrometry method was developed and validated to disclose the influence of stilbene glucoside on the pharmacokinetics of emodin in rats. Drug and Statistics 2.0 was used for the estimation of the pharmacokinetic parameters. Gene expression analysis in liver and intestinal tissues was performed by a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) method. RESULTS: The analytical method appeared to be suitable for the analysis of emodin with desirable linearity, accuracy, precision and stability, and the total analysis time was less than 2 min on a short column. Glucuronide of emodin, which is the major metabolite of emodin, was determined after ß-glucuronidase hydrolysis. As the in vivo pharmacokinetic studies had indicated, the AUC, Cmax and T1/2 of emodin were increased after the stilbene glucoside treatment, and the glucuronidation of emodin was significantly inhibited. The mRNA levels from UGT1A8 and UGT1A2 were decreased by stilbene glucoside treatment. In contrast, the expression of UGT1A1, UGT1A6 and UGT1A9 mRNA was increased in the liver following treatment. CONCLUSIONS: The influence of stilbene glucoside on the pharmacokinetics of emodin may be attributed to the inhibition of UGT1A8 mRNA expression. Thus, it is important to extend this research to deepen our understanding of the pharmacological and/or toxicological effects of RPM.