Targeting erythrocyte-mediated hypoxia to alleviate lung injury induced by pyrrolizidine alkaloids.

Pyrrolizidine alkaloids (PAs) are widely distributed natural toxins and have been extensively studied for their hepatotoxicity. However, PA-induced pulmonary toxicity remains less studied regarding the initiating mechanism and treatment approaches. Our previous study demonstrated the formation of pyrrole-hemoglobin adducts after PA exposure in vivo, which is suspected to affect the oxygen-carrying capacity of erythrocytes [red blood cells (RBCs)] consequently. The present study aimed to investigate the effects of PAs on the oxygen-carrying capacity of RBCs and the potential of targeting RBC-mediated hypoxia to alleviate PA-induced lung injury. First, rats were treated with retrorsine (RTS) or monocrotaline (MCT) intravenously at 0.2 mmol/kg. The results of Raman spectrometry analysis on blood samples revealed both RTS and MCT significantly reduced the oxygen-carrying capacity of RBCs. Further, MCT (0.2 mmol/kg) was orally given to the rats with or without pretreatment with two doses of erythropoietin (Epo, 500 IU/kg/dose every other day), an RBC-stimulating agent. Biochemical and histological results showed pretreatment with Epo effectively reduced the cardiopulmonary toxicity induced by MCT. These findings provide the first evidence that adduction on hemoglobin, and the resulting RBC damage and impaired oxygen-carrying capacity, are the major initiating mechanism underlying PA-induced pulmonary arterial hypertension (PAH), while targeting the RBC damage is a potential therapeutic approach for PA-induced lung injury.

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.

Correlation Investigation between Pyrrole-DNA and Pyrrole-Protein Adducts in Male ICR Mice Exposed to Retrorsine, a Hepatotoxic Pyrrolizidine Alkaloid.

Pyrrolizidine alkaloids (PAs) have been found in over 6000 plants worldwide and represent the most common hepatotoxic phytotoxins. Catalyzed by hepatic cytochrome P450 enzymes, PAs are metabolized into reactive pyrrolic metabolites, which can alkylate cellular proteins and DNA to form pyrrole-protein adducts and pyrrole-DNA adducts, leading to cytotoxicity, genotoxicity, and tumorigenicity. To date, the correlation between these PA-derived pyrrole-protein and pyrrole-DNA adducts has not been well investigated. Retrorsine is a representative hepatotoxic and carcinogenic PA. In the present study, the correlations among the PA-derived liver DNA adducts, liver protein adducts, and serum protein adducts in retrorsine-treated mice under different dosage regimens were studied. The results showed positive correlations among these adducts, in which serum pyrrole-protein adducts were more accessible and present in higher abundance, and thus could be used as a suitable surrogate biomarker for pyrrole-DNA adducts to indicate the genetic or carcinogenic risk posed by retrorsine.

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.

Fasting augments pyrrolizidine alkaloid-induced hepatotoxicity.

Pyrrolizidine alkaloids (PAs) are phytotoxins widely present in various natural products and foodstuffs. The present study aims to investigate the effects of fasting on PA-induced hepatotoxicity and the underlying biochemical mechanisms. The results of hepatotoxic study showed that 15-h overnight fasting significantly exacerbated the hepatotoxicity of retrorsine (RTS, a representative toxic PA) in fasted rats compared to fed rats, as indicated by remarkably elevated plasma ALT and bilirubin levels and obvious liver histological changes. Further toxicokinetic studies revealed that fasting significantly enhanced cytochromes P450 enzymes (CYPs)-mediated metabolic activation of RTS leading to increased formation of pyrrole-protein adducts and thus decreased the in vivo exposure and excretion of both parent RTS and its N-oxide metabolite. Metabolic studies demonstrated that fasting induced enzyme activities of CYP1A2, CYP2B6 and CYP2E1 that participated in catalyzing RTS to its reactive pyrrolic metabolites. Moreover, fasting also dramatically decreased hepatic glutathione (GSH) content, which restricted the detoxification of GSH by neutralizing the reactive pyrrolic metabolite of RTS, further contributing to the enhanced hepatotoxicity. The present findings may have an impact on future PA toxicity tests with different dietary styles and/or risk assessment of metabolite-mediated toxins by considering the profound effects of fasting.

Association of MALAT-1 gene single nucleotide polymorphisms with genetic susceptibility to systemic lupus erythematosus.

Background: Abnormal expression and function of long non-coding RNAs (lncRNAs) are closely related to the pathogenesis of systemic lupus erythematosus (SLE). In this study, we aimed to investigate the association of lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT-1) gene single-nucleotide polymorphisms (SNPs) with susceptibility and clinical characteristics of SLE patients. Methods: A case-control study including 489 SLE patients and 492 healthy controls was conducted. Four MALAT-1 SNPs (rs4102217, rs591291, rs11227209, and rs619586) were genotyped in all subjects, their correlation with SLE susceptibility and clinical characteristics were also analyzed. Results: Results showed that the rs4102217 locus was associated with the risk of SLE. In recessive models, the GG+CG genotype of rs4102217 was associated with the decreased risk of SLE compared to CC (p = 0.036, OR = 0.348, 95% CI: 0.124-0.975). In additive models, the GG genotype of rs4102217 was associated with the decreased risk of SLE compared to CC (p = 0.040, OR = 0.355, 95% CI: 0.127-0.996). However, no association was found between MALAT-1 gene polymorphism and clinical manifestations of SLE (all p > 0.05). Conclusion: In summary, MALAT-1 rs4102217 is associated with susceptibility to SLE, suggesting that MALAT-1 may play a role in SLE.

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.

Mutational Signature Analysis Reveals Widespread Contribution of Pyrrolizidine Alkaloid Exposure to Human Liver Cancer.

BACKGROUND AND AIMS: Mutational signature analyses are an effective tool in identifying cancer etiology. Humans are frequently exposed to pyrrolizidine alkaloids (PAs), the most common carcinogenic phytotoxins widely distributed in herbal remedies and foods. However, due to the lack of human epidemiological data, PAs are classified as group II hepatocarcinogens by the World Health Organization. This study identified a PA mutational signature as the biomarker to investigate the association of PA exposure with human liver cancer. APPROACH AND RESULTS: Pyrrole-protein adducts (PPAs), the PA exposure biomarker, were measured and found in 32% of surgically resected specimens from 34 patients with liver cancer in Hong Kong. Next, we delineated the mode of mutagenic and tumorigenic actions of retrorsine, a representative PA, in mice and human hepatocytes (HepaRG). Retrorsine induced DNA adduction, DNA damage, and activation of tumorigenic hepatic progenitor cells, which initiated hepatocarcinogenesis. PA mutational signature, as the unique molecular fingerprint of PA-induced mutation, was derived from exome mutations in retrorsine-exposed mice and HepaRG cells. Notably, PA mutational signature was validated in genomes of patients with PPA-positive liver cancer but not patients with PPA-negative liver cancer, confirming the specificity of this biomarker in revealing PA-associated liver cancers. Furthermore, we examined the established PA mutational signature in 1,513 liver cancer genomes and found that PA-associated liver cancers were potentially prevalent in Asia (Mainland China [48%], Hong Kong [44%], Japan [22%], South Korea [6%], Southeast Asia [25%]) but minor in Western countries (North America [3%] and Europe [5%]). CONCLUSIONS: This study provides a clinical indication of PA-associated liver cancer. We discovered an unexpectedly extensive implication of PA exposure in patients with liver cancer, laying the scientific basis for precautionary approaches and prevention of PA-associated human liver cancers.

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.

Emerging role of Fli1 in autoimmune diseases.

The Ets transcription factor family exerts crucial role in cell proliferation, apoptosis, differentiation and migration. Friend leukemia integration 1 (Fli1), a member of the Ets family, is expressed in fibroblasts, endothelial cells and immune cells. Fli1 gene is participated in the development, proliferation, activation, migration and other processes of immune cells. Fli1 can also affect the function of immune cells by regulating cytokines and chemokines. Emerging evidence has shown that Fli1 is implicated in the etiology of several autoimmune diseases, including systemic sclerosis (SSc) and systemic lupus erythematosus (SLE). In this review, we mainly discuss the current evidence for the role of Fli1 in these diseases.

Lung injury induced by pyrrolizidine alkaloids depends on metabolism by hepatic cytochrome P450s and blood transport of reactive metabolites.

Pyrrolizidine alkaloids (PAs) are common phytotoxins with both hepatotoxicity and pneumotoxicity. Hepatic cytochrome P450 enzymes are known to bioactivate PAs into reactive metabolites, which can interact with proteins to form pyrrole-protein adducts and cause intrahepatic cytotoxicity. However, the metabolic and initiation biochemical mechanisms underlying PA-induced pneumotoxicity remain unclear. To investigate the in vivo metabolism basis for PA-induced lung injury, this study used mice with conditional deletion of the cytochrome P450 reductase (Cpr) gene and resultant tissue-selective ablation of microsomal P450 enzyme activities. After oral exposure to monocrotaline (MCT), a pneumotoxic PA widely used to establish animal lung injury models, liver-specific Cpr-null (LCN) mice, but not extrahepatic Cpr-low (xh-CL) mice, had significantly lower level of pyrrole-protein adducts in the serum, liver and lungs compared with wild-type (WT) mice. While MCT-exposed LCN mice had significantly higher blood concentration of intact MCT, compared to MCT-exposed WT or xh-CL mice. Consistent with the MCT in vivo bioactivation data, MCT-induced lung injury, represented by vasculature damage, in WT and xh-CL mice but not LCN mice. Furthermore, reactive metabolites of MCT were confirmed to exist in the blood efflux from the hepatic veins of MCT-exposed rats. Our results provide the first mode-of-action evidence that hepatic P450s are essential for the bioactivation of MCT, and blood circulating reactive metabolites of MCT to the lung causes pneumotoxicity. Collectively, this study presents the scientific basis for the application of MCT in animal lung injury models, and more importantly, warrants public awareness and further investigations of lung diseases associated with exposure to not only MCT but also different PAs.

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.

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.

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.

Paeoniflorin protects against lipopolysaccharide-induced acute lung injury in mice by alleviating inflammatory cell infiltration and microvascular permeability.

OBJECTIVE: The present study aims to explore the effects of paeoniflorin (PF), a monoterpene glycoside isolated from the roots of Paeonia lactiflora Pallas, on acute lung injury (ALI) and the possible mechanisms. MATERIALS AND METHOD: ALI was induced in mice by an intratracheal instillation of lipopolysaccharide (LPS, 1 mg/kg), and PF was injected intraperitoneally 30 min prior to LPS administration. After 24 h, lung water content, histology, microvascular permeability and proinflammatory cytokines in the bronchoaveolar lavage fluid were evaluated. RESULTS: It was shown that PF (50, 100 mg/kg) could alleviate LPS-induced ALI, evidenced by reduced pulmonary edema, improved histological changes, and attenuated inflammatory cell accumulation in the interstitium and alveolar space as well as microvascular permeability. It also markedly down-regulated the expressions of proinflammatory cytokines interleukin (IL)-1ß and tumor necrosis factor (TNF)-α at both transcription and protein levels. Additionally, PF inhibited the phosphorylations of p38 MAP kinase (p38) and c-Jun NH2-terminal kinase (JNK) but not extracellular signal-regulated kinase (ERK), and prevented the activation of nuclear factor-kappa B (NF-κB) in the lung tissues. CONCLUSION: The findings suggest that PF is able to alleviate ALI, and the underlying mechanisms are probably attributed to decreasing the production of proinflammatory cytokines through down-regulation of the activation of p38, JNK and NF-κB pathways in lung tissues.