Pyrrole-protein Adducts Research Articles

In vitro-in silico study on the influence of dose, fraction bioactivated and endpoint used on the relative potency value of pyrrolizidine alkaloid N-oxides compared to parent pyrrolizidine alkaloids

Pyrrolizidine alkaloids (PAs) and their N-oxides (PA-N-oxides) are phytotoxins found in food, feed and the environment. Yet, limited data exist from which the relative potency of a PA-N-oxide relative to its corresponding PA (REPPANO to PA) can be defined. This study aims to investigate the influence of dose, fraction bioactivated and endpoint on the REPPANO to PA of a series of pyrrolizidine N-oxides using in vitro-in silico data and physiologically based kinetic (PBK) modeling. The first endpoint used to calculate the REPPANO to PA was the ratio of the area under the concentration–time curve of PA resulting from an oral dose of PA-N-oxide divided by that from an equimolar dose of PA (Method 1). The second endpoint was the ratio of the amount of pyrrole-protein adducts formed under these conditions (Method 2). REPPANO to PA values appeared to decrease with increasing dose, with the decrease for Method 2 already starting at lower dose level than for Method 1. At dose levels as low as estimated daily human intakes, REPPANO to PA values amounted to 0.92, 0.81, 0.78, and 0.68 for retrorsine N-oxide, seneciphylline N-oxide, riddelliine N-oxide and senecivernine N-oxide, respectively, and became independent of the dose or fraction bioactivated, because no GSH depletion, saturation of PA clearance or PA-N-oxide reduction occurs. Overall, the results demonstrate the strength of using PBK modeling in defining REPPANO to PA values, thereby substantiating the use of the same approach for other PA-N-oxides for which in vivo data are lacking.

The toxicokinetic and metabolism of structurally diverse pyrrolizidine alkaloids in rats

Ethnopharmacological relevancePyrrolizidine 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 studyThis 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 methodsThe 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. ResultsMCT 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. ConclusionDramatic 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.

Pyrrolizidine plant extracts as diagnostic biochemical markers on therapeutic consequences

The consumption of cereals and bakery products contaminated with seeds of species containing Pyrrolizidine alkaloids (PAs) has been involved in mass poisonings in rural areas of Afghanistan, India, South Africa and the former USSR. Poisoning can manifest as acute or subacute veno-occlusive disease with specific symptoms such as persistent hepatomegaly, leading to cirrhosis or shown to have genotoxic, mutagenic, teratogenic and carcinogenic effects. Several such toxicity testing of PAs have been done in cultured animal cells with reference to mice model, to better understand the pathway of metabolic impairement. Also the pluripotent proprty of mice bone marrow cells show better analyzing capacity of differentiated cell lineages. Therefore this pre-analytic review aims to focus the risk evaluation methods of PA exposure on current knowledge and recent advances of metabolic pathways of different PAs. In addition, this review provides perspectives of precision toxicity assessment strategies and biomarker development for the risk control of human intoxication by PAs with eventual categorization of pyrrole-protein adducts like serum Alanine Transferase (ALT), Glutathione transferase (GSH-T), Alkaline Phosphatase (ALP) in different cell lineages grown by lab-based cell culture techniques.

Physiologically based kinetic modeling of senecionine N-oxide in rats as a new approach methodology to define the effects of dose and endpoint used on relative potency values of pyrrolizidine alkaloid N-oxides.

Over 1,000 pyrrolizidine alkaloids (PAs) and their N-oxides (PA-N-oxides) occur in 3% of all flowering plants. PA-N-oxides are toxic when reduced to their parent PAs, which are bioactivated into pyrrole intermediates that generate protein- and DNA-adducts resulting in liver toxicity and carcinogenicity. Literature data for senecionine N-oxide in rats indicate that the relative potency (REP) value of this PA-N-oxide compared to its parent PA senecionine varies with the endpoint used. The first endpoint was the ratio between the area under the concentration-time curve (AUC) for senecionine upon dosing senecionine N-oxide or an equimolar dose of senecionine, while the second endpoint was the ratio between the amount for pyrrole-protein adducts formed under these conditions. This study aimed to investigate the mode of action underlying this endpoint dependent REP value for senecionine N-oxide with physiologically based kinetic (PBK) modeling. Results obtained reveal that limitation of 7-GS-DHP adduct formation due to GSH depletion, resulting in increased pyrrole-protein adduct formation, occurs more likely upon high dose oral PA administration than upon an equimolar dose of PA-N-oxide. At high dose levels, this results in a lower REP value when based on pyrrole-protein adduct levels than when based on PA concentrations. At low dose levels, the difference no longer exists. Altogether, the results of the study show how the REP value for senecionine N-oxide depends on dose and endpoint used, and that PBK modeling provides a way to characterize REP values for PA-N-oxides at realistic low dietary exposure levels, thus reducing the need for animal experiments.

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.

Firm evidence for the detoxification of senecionine-induced hepatotoxicity via N-glucuronidation in UGT1A4–humanized transgenic mice

Uridine diphosphate glucuronosyltransferase (UGT)1A4 is responsible for N-glucuronidation of tertiary amines but is a pseudogene in commonly used rodent models in toxicity and safety assessment. As a continuation of our investigation into the toxicity and safety assessment of pyrrolizidine alkaloid (PA)-containing herbs, we generated a UGT1A4–humanized (hUGT1A4) transgenic mouse model to systematically study the toxicity, metabolism network, and toxicokinetic characteristics of senecionine (a representative toxic PA) and compared with that in the wide-type controls in parallel. As results, senecionine-induced toxicity was significantly decreased as approved by mortality, pathology, and biochemistry assays in hUGT1A4 mice and cultured primary hepatocytes. More importantly N-glucuronidation adduct was exclusively identified in all the hUGT1A4 mice, liver microsomes, and cultured primary hepatocytes, yet absent in the wide-type controls. The variation in toxicokinetic characters was also observed between hUGT1A4 mice and the wide-type controls with a notably inhibition of the toxification metabolites, i.e., pyrrole-protein adducts, in hUGT1A4 mice. Conclusively, UGT1A4 plays an important role in detoxification of senecionine and the hUGT1A4 mouse model is promising for the pre-clinical evaluation of the efficacy and toxicity of tertiary amine agents in drug development and safety assessment.

Bear bile powder attenuates senecionine-induced hepatic sinusoidal obstruction syndrome in mice.

Hepatic sinusoidal obstruction syndrome (HSOS) via exposure to pyrrolizidine alkaloids (PAs) is with high mortality and there is no effective treatment in clinics. Bear bile powder (BBP) is a famous traditional animal drug for curing a variety of hepatobiliary diseases such as cholestasis, inflammation, and fibrosis. Here, we aim to evaluate the protective effect of BBP against HSOS induced by senecionine, a highly hepatotoxic PA compound. Our results showed that BBP treatment protected mice from senecionine-induced HSOS dose-dependently, which was evident by improved liver histology including reduced infiltration of inflammatory cells and collagen positive cells, alleviated intrahepatic hemorrhage and hepatic sinusoidal endothelial cells, as well as decreased conventional serum liver function indicators. In addition, BBP treatment lowered matrix metalloproteinase 9 and pyrrole-protein adducts, two well-known markers positively associated with the severity of PA-induced HSOS. Further investigation showed that BBP treatment prevents the development of liver fibrosis by decreasing transforming growth factor beta and downstream fibrotic molecules. BBP treatment also alleviated senecionine-induced liver inflammation and lowered the pro-inflammatory cytokines, in which tauroursodeoxycholic acid played an important role. What's more, BBP treatment also decreased the accumulation of hydrophobic bile acids, such as cholic acid, taurocholic acid, glycocholic acid, as well. We concluded that BBP attenuates senecionine-induced HSOS in mice by repairing the bile acids homeostasis, preventing liver fibrosis, and alleviating liver inflammation. Our present study helps to pave the way to therapeutic approaches of the treatment of PA-induced liver injury in clinics.

Liquorice Extract and 18β-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 18β-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.

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.

Diurnal hepatic CYP3A11 contributes to chronotoxicity of the pyrrolizidine alkaloid retrorsine in mice

1. Retrorsine (RTS) is a pyrrolizidine alkaloid (distributed in many medicinal plants) that has significant hepatotoxicity. Here, we aimed to determine the daily variations in RTS hepatotoxicity (chronotoxicity) in mice, and to investigate the role of metabolism in generating RTS chronotoxicity. 2. Acute toxicity and pharmacokinetic studies were performed with mice after RTS administration at different times of the day. Hepatotoxicity was assessed by measuring plasma ALT (alanine aminotransferase) and AST (aspartate aminotransferase) levels. mRNA and proteins were determined by qPCR and Western blotting, respectively. Time-dependent in vitro metabolism of RTS was assessed by using mouse liver microsomes. 3. We found that RTS toxicity was more severe in the dark phase (zeitgeber time 14 or ZT14 and ZT18) than in the light phase (ZT2 and ZT6). This chronotoxicity was associated with a dosing time difference in the systemic exposures of RTS and a pyrrolic ester metabolite (a cause of hepatotoxicity, measured by the levels of pyrrole-GSH conjugate and pyrrole-protein adducts due to a high chemical reactivity). Moreover, the CYP3A11 (a major enzyme for RTS bioactivation) inhibitor ketoconazole decreased the production of pyrrole-GSH conjugate and abrogated diurnal rhythm in RTS metabolism. In addition, E4bp4 (a circadian regulator of Cyp3a11) ablation abolished the rhythm of CYP3A11 expression and abrogated the dosing time-dependency of RTS toxicity. 4. In conclusion, RTS chronotoxicity in mice was attributed to time-varying hepatic metabolism regulated by the circadian clock. Our findings have implications for reducing pyrrolizidine alkaloid-induced toxicity via a chronotherapeutic approach.

Metabolism-mediated cytotoxicity and genotoxicity of pyrrolizidine alkaloids.

Pyrrolizidine alkaloids (PAs) and PA N-oxides are common phytotoxins produced by over 6000 plant species. Humans are frequently exposed to PAs via ingestion of PA-containing herbal products or PA-contaminated foods. PAs require metabolic activation to form pyrrole-protein adducts and pyrrole-DNA adducts which lead to cytotoxicity and genotoxicity. Individual PAs differ in their metabolic activation patterns, which may cause significant difference in toxic potency of different PAs. This review discusses the current knowledge and recent advances of metabolic pathways of different PAs, especially the metabolic activation and metabolism-mediated cytotoxicity and genotoxicity, and the risk evaluation methods of PA exposure. In addition, this review provides perspectives of precision toxicity assessment strategies and biomarker development for the risk control and translational investigations of human intoxication by PAs.

The first evidence of pyrrolizidine alkaloid‐associated human liver cancer

Pyrrolizidine alkaloids (PAs) are one of the most common phytotoxins with liver mutagenicity and tumorigenicity. Humans are frequently exposed to PAs via the ingestion of PA-containing herbal remedies and PA-contaminated foods. However, due to the lack of human epidemiological data, whether PA exposure is correlated with human liver cancer is unexplored. Here, we developed PA mutational signature as the biomarker to investigate the association of PA exposure in human liver cancer. Firstly, pyrrole-protein adducts (PPA), a PA exposure biomarker, were measured and found for the first time in 32% of 34 liver cancer patients in Hong Kong. Next, we delineated mode of mutagenic and tumorigenic actions of retrorsine, a representative PA, in mice. PA mutational signature, the imprint of PA-induced mutations with liver cancer relevance, was derived by analyzing exome mutations in mice with retrorsine-induced liver tumor, followed by specificity validation in clinical PPA-positive samples. Further, we examined and found that the established PA mutational signature was implicated in 44% of public liver cancer genomes in Hong Kong. Our findings provide the first evidence of PA exposure in liver cancer patients. By utilizing the newly identified PA mutational signature, we discover an unexpectedly extensive implication of PA exposure in liver cancer patients, laying a scientific basis for future prevention and precautionary approaches of PA-associated human liver cancers.

Metabolic Activation of Retrorsine may Disrupt Bile Acid Homeostasis in Mice through the Nrf2 Pathway

The hepatotoxic pyrrolizidine alkaloids (PAs) were reported to increase bile acid (BA) levels in the rat. However, it is still unclear whether the production of highly reactive dehydropyrrolizidine through CYP450s is directly relevant to BA changes. To further explore the mechanism by which metabolic activation of PAs induced BA changes, the effect of impaired or enhanced metabolic activation on the BA profiling and BA-related synthesis and to investigate transport genes, and explore the involvement of the Nrf2 pathway. Blood and liver samples were collected after intragastrical administration of 35 mg/kg retrorsine or saline for seven days in wild-type (WT) and Nrf2 KO mice. CYP450 inhibitor, 1-aminobenzotriazole (ABT), or gammaglutamylcysteine synthetase inhibitor, L-buthionine-sulfoximine (BSO) were employed in WT mice. Retrorsineinduced hepatotoxicity was evaluated by a biochemical method and H&E staining method. Serum BAs were quantified by high-performance liquid chromatography/triple quadrupole mass spectrometry. Blood pyrrole-protein adducts were semi quantified by high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry. The gene and protein expression of BA-related transporters and enzymes in the liver were measured by a quantitative real-time PCR method and western blotting method. The BA concentrations in serum were increased in the retrorsine-treated WT mice, along with the upregulation of BA transporters, Ostβ, Mrp3, Mrp4, and Mrp2. When ABT was co-administered, the altered BA levels and Mrp4 mRNA and protein levels were reversed, accompanied by a 50% reduction of 6,7-dihydro-7-hydroxy-1- hydroxymethyl-5H-pyrrolizine (DHP) formation. When BSO was co-administered, serum BAs were not further increased, but Ostβ, Mrp3, Mrp4 mRNA, and Mrp4 protein levels continuously increased. The induction of Mrp4 by retrorsine among the tested BA transporters was the only one that was abolished or enhanced in the presence of ABT or BSO. The Nrf2 protein levels in the nucleus increased in the retrorsine-treated WT mice, which were remarkably repressed by co-administration of ABT and enhanced by co-administration of BSO. In Nrf2 KO mice receiving retrorsine, the bile acids and the mRNA and protein levels of Mrp2, Mrp3, Mrp4, and Ostβ were hardly changed, indicating the direct role of Nrf2 in retrorsine-induced BA changes in WT mice. The activation of Nrf2 translocation by forming the reactive metabolite of PAs induced the expressions of BA transporters and changed serum BA levels. Mrp4 was a sensitive biomarker for the perturbation of redox status caused by the formation of dehydropyrrolizidine.

Clinical application of pyrrole-hemoglobin adducts as a biomarker of pyrrolizidine alkaloid exposure in humans.

Pyrrolizidine alkaloids (PAs) are naturally occurring hepatotoxins widely present in hundreds of plant species and also known to contaminate many foodstuffs, such as grain, honey, and tea. The formation of pyrrole-protein adducts via metabolic activation of PAs has been suggested as a primary trigger initiating hepatotoxicity. The present study for the first time tested the suitability of pyrrole-hemoglobin adducts as a novel and specific biomarker of PA exposure in humans. The level and elimination kinetics of pyrrole-hemoglobin adducts were systematically investigated in the blood samples of 43 PA-induced liver injury (PA-ILI) patients. The results revealed significantly higher concentrations (84.50 ± 78.38nM) and longer persistence (~ 4months) of pyrrole-hemoglobin adducts than that (concentration: 9.53 ± 10.72nM; persistence: ~ 2months) of pyrrole-plasma protein adducts, our previously developed PA exposure biomarker. Our findings confirmed that pyrrole-hemoglobin adducts with higher level and longer persistence should serve as a more applicable PA exposure biomarker for future clinical diagnosis of PA-ILI in drug/herb-induced liver injury patients.

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

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. 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. 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. 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. 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.

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.

Protein cross-linking in primary cultured mouse hepatocytes by dehydropyrrolizidine alkaloids: Structure–toxicity relationship

Pyrrolizidine alkaloids (PAs) are natural toxins found in about 3%–5% of flowering plants. Dehydropyrrolizidine alkaloids contain a double bond in 1, 2-position of the necine bases, including retronecine type PAs (RET-PAs) and their N-oxides (RET N-oxide-PAs), and otonecine type PAs (OTO-PAs), and are known for their significant hepatotoxicity. Most dehydropyrrolizidine alkaloids are metabolically activated by cytochrome P450 (CYP450) enzymes to generate active pyrroles, which further bind to proteins to form pyrrole–protein adducts (PPAs). Methods for predicting PA-induced liver injury are generally performed on in vitro models with extremely low activities of CYP450 enzymes, which is different from the situation in vivo. In this regard, primary cultured mouse hepatocytes, which showed comparable CYP450 activity with the in vivo models, were applied to illustrate the structure–toxicity relationship of 13 dehydropyrrolizidine alkaloids, namely, eight RET-PAs, three RET N-oxide-PAs, and two OTO-PAs. PA-induced cytotoxicity and PA-generated PPAs were analyzed in primary mouse hepatocytes treated with different PAs. Results showed that PA-induced toxicity was correlated with the amount of PA-generated PPAs. RET-PAs and OTO-PAs were generally more toxic than RET N-oxide-PAs and generated higher amount of PPAs. PPAs were utilized to evaluate the efficiency of metabolic activation and predict the toxic potencies of dehydropyrrolizidine alkaloids. The proposed model could be a new approach for toxicity evaluation and risk control of exposure to 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.

Establishment of a novel CYP3A4-transduced human hepatic sinusoidal endothelial cell model and its application in screening hepatotoxicity of pyrrolizidine alkaloids

Pyrrolizidine alkaloids (PAs) are extensively distributed in plants and are known to damage hepatic sinusoidal endothelial cells (HSECs) via metabolic activation mediated by hepatic cytochrome P450 enzymes (CYPs), particularly the CYP3A4 isozyme. Different PAs have distinct toxic potencies and their toxic effects on HSECs are difficult to be determined in cultured cells, because HSECs lack the key CYP3A4 isozyme for metabolic activation. This study aims to establish a novel, convenient and reliable CYP3A4-expressing HSEC model using human HSECs transduced with lentivirus carrying CYP3A4-ires-eGFP, for evaluating the hepatotoxicity of different PAs on their target HSECs. The developed CYP3A4-expressing HSEC (HSEC-CYP3A4) model was verified by the expression of GFP and CYP3A4 and by the ability to metabolize nifedipine, a classic CYP3A4 substrate. Treated with retrorsine, a representative toxic PA, HSEC-CYP3A4 cells showed significantly reduced cell viability, depletion of GSH, and increased formation of pyrrole-protein adducts. Furthermore, this newly developed cell model successfully discriminated the cytotoxic potency of different PAs evidenced by their IC40 values. In conclusion, the established HSEC-CYP3A4 cell model can be used as a rapid screening platform for assessing the relative potencies of individual PAs on their target HSECs and for investigating the mechanisms underlying PA-induced hepatic sinusoidal damage.

Investigation on Pneumotoxicity Induced by Pyrrolizidine AlkaloidsInvestigation on Pneumotoxicity Induced by Pyrrolizidine Alkaloids

Pyrrolizidine alkaloids (PAs) are most common phytotoxins. Apart from their extensively reported hepatotoxicity, few PAs, such as MCT, are also reported to cause lung injury. However, whether different PAs commonly lead to pneumotoxicity remains largely unknown whether different PAs. PAs require metabolic activation to exert toxicity. Hepatic cytochrome P450s are proposed to be responsible for bioactivation of PAs to reactive dehydro‐PAs, which interact with proteins to form pyrrole‐protein adducts causing intrahepatic cytotoxicity. However, the mechanisms underlying pneumotoxicity of PAs, especially the site of metabolic activation of PAs, have not been delineated. Here we aimed to investigate pneumotoxicity of different types of PAs and also identify the contribution of hepatic P450s and extrahepatic P450s to metabolic bioactivation of PAs.Pulmonary toxicity of four representative PAs of both retronecine and otonecine types and Gynura Segetum (GS) alkaloid extract containing two retronecine type PAs, to mimic human PA poisoning, were investigated. Rats were orally administered with representative PAs and GS at 0.2 mmol/kg for 48 hours. Biochemical and histological examinations were applied to evaluate PA‐induced toxicity in liver and lung. The results showed that all PAs induced liver injury to varied levels. Significant lung lesions including pulmonary arterial hypertrophy, capillary congestion, endothelium damage, and abnormal proliferation of smooth muscle cells were found in all PA‐treated groups, demonstrating that both retronecine and otonecine types of PAs can cause pulmonary toxicity.Further to delineate the role of hepatic and extrahepatic CYPs in metabolic bioactivation of PAs, mice with conditional deletion of cytochrome P450 reductase (Cpr) gene and resultant dysfunctional P450s were used. After oral exposure to MCT (120 mg/kg), a well‐known pneumotoxic PA, metabolic activation and pneumotoxicity of MCT among wild‐type (WT), liver‐specific Cpr‐null (LCN), and extrahepatic Cpr‐knockdown (XhCL) mice were compared. The results demonstrated that comparing with WT mice, pyrrole‐protein adducts were significantly decreased (by >60%) in the serum, liver and lungs in LCN mice but unchanged in XhCL mice. In addition, comparing with WT mice, MCT‐exposed LCN mice had significantly higher blood concentration of the intact MCT. Consistently, lung injury represented by vasculature damage was observed in WT and XhCL mice but not in LCN mice. All the data confirmed that hepatic CYPs, not extrahepatic CYPs, were responsible for metabolic activation of PAs.To conclude, the present study for the first time demonstrated that PAs can commonly induce lung injury, which is dependent on the metabolic activation mediated by functional hepatic CYPs.Support or Funding InformationThe present study was supported by General Research Fund (GRF Project No. 14160817) from Research Grants Council of Hong Kong Special Administrative Region.