Hepatotoxicity screening and ranking of structurally different pyrrolizidine alkaloids in zebrafish.

Pyrrolizidine alkaloids (PAs) are phytotoxins distributed in ∼6000 plant species. PA-contaminated/containing foodstuffs/herbs/supplements pose a potential threat to human health. Various regulatory authorities established different PA margins of exposure assuming an equal hepatotoxic potency of structurally diverse PAs, although they exhibit different toxic potencies. Therefore, understanding hepatotoxic potencies of different PAs would facilitate a more appropriate risk assessment of PA exposure. In this study, a zebrafish model, which mimics physiological processes of absorption, distribution, metabolism, and excretion, was selected to evaluate acute hepatotoxic potency of different PAs (7 PAs and 2 PA N-oxides) and explore possible physiological pathways involved in PA-induced hepatotoxicity. After 6 h oral administration, PAs caused distinct structure-dependent hepatotoxicity with a series of biochemical and histological changes in zebrafish. Based on the measured toxicological endpoints, the relative toxic potency order of different PAs was derived as lasiocarpine âˆ¼ retrorsine > monocrotaline > riddelliine > clivorine > heliotrine > retrorsine N-oxide âˆ¼ riddelliine N-oxide≫>platyphyline. Further, compared to control group, different upregulation/downregulation of mRNA expression in PA-treated groups indicated that inflammation, apoptosis, and steatosis were involved in PA-induced hepatotoxicity in zebrafish. These findings demonstrate that zebrafish model is useful for screening and ranking hepatotoxicity of PAs with diverse structures, which would facilitate the more accurate risk assessment of PA exposure.

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.

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.

Novel Insights into Pyrrolizidine Alkaloid Toxicity and Implications for Risk Assessment: Occurrence, Genotoxicity, Toxicokinetics, Risk Assessment-A Workshop Report.

This paper reports on the major contributions and results of the 2nd International Workshop of Pyrrolizidine Alkaloids held in September 2020 in Kaiserslautern, Germany. Pyrrolizidine alkaloids are among the most relevant plant toxins contaminating food, feed, and medicinal products of plant origin. Hundreds of PA congeners with widespread occurrence are known, and thousands of plants are assumed to contain PAs. Due to certain PAs' pronounced liver toxicity and carcinogenicity, their occurrence in food, feed, and phytomedicines has raised serious human health concerns. This is particularly true for herbal teas, certain food supplements, honey, and certain phytomedicinal drugs. Due to the limited availability of animal data, broader use of in vitro data appears warranted to improve the risk assessment of a large number of relevant, 1,2-unsaturated PAs. This is true, for example, for the derivation of both toxicokinetic and toxicodynamic data. These efforts aim to understand better the modes of action, uptake, metabolism, elimination, toxicity, and genotoxicity of PAs to enable a detailed dose-response analysis and ultimately quantify differing toxic potencies between relevant PAs. Accordingly, risk-limiting measures comprising production, marketing, and regulation of food, feed, and medicinal products are discussed.

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.

Machine Learning for Predicting Risk of Drug-Induced Autoimmune Diseases by Structural Alerts and Daily Dose.

An effective approach for assessing a drug's potential to induce autoimmune diseases (ADs) is needed in drug development. Here, we aim to develop a workflow to examine the association between structural alerts and drugs-induced ADs to improve toxicological prescreening tools. Considering reactive metabolite (RM) formation as a well-documented mechanism for drug-induced ADs, we investigated whether the presence of certain RM-related structural alerts was predictive for the risk of drug-induced AD. We constructed a database containing 171 RM-related structural alerts, generated a dataset of 407 AD- and non-AD-associated drugs, and performed statistical analysis. The nitrogen-containing benzene substituent alerts were found to be significantly associated with the risk of drug-induced ADs (odds ratio = 2.95, p = 0.0036). Furthermore, we developed a machine-learning-based predictive model by using daily dose and nitrogen-containing benzene substituent alerts as the top inputs and achieved the predictive performance of area under curve (AUC) of 70%. Additionally, we confirmed the reactivity of the nitrogen-containing benzene substituent aniline and related metabolites using quantum chemistry analysis and explored the underlying mechanisms. These identified structural alerts could be helpful in identifying drug candidates that carry a potential risk of drug-induced ADs to improve their safety profiles.

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.

Metabolism of carcinogenic pyrrolizidine alkaloids and pyrrolizidine alkaloid N-oxides by rat primary hepatocytes generate the same characteristic DHP-DNA adducts.

We recently established a genotoxic mechanism mediated by a set of (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-DNA adducts, which lead to pyrrolizidine alkaloid (PA)-induced liver tumor initiation. This mechanism is involved in the metabolism of a series of carcinogenic PAs and PA N-oxides in rats in vivo and in vitro. There is a correlation between the order of liver tumor potency and the level of DHP-DNA adduct formation. Thus, these DHP-DNA adducts can be potential biomarkers of PA and PA N-oxide exposure and liver tumor initiation. To establish the generality of this mechanism, in the present study, we examined the metabolism of 13 potential carcinogenic PAs, 1 non-carcinogenic PA, and 5 PA N-oxides by male rat primary hepatocytes. With the exception of the nontoxic PA and vehicle control, all treated groups produced identical set of DHP-DNA adducts. These results support a general genotoxic mechanism mediated by the formation of characteristic DHP-DNA adducts leading to PA-induced liver tumor initiation.

Applications of electron spin resonance spectroscopy in photoinduced nanomaterial charge separation and reactive oxygen species generation.

Nano-metals, nano-metal oxides, and carbon-based nanomaterials exhibit superior solar-to-chemical/photo-electron transfer properties and are potential candidates for environmental remediations and energy transfer. Recent research effort focuses on enhancing the efficiency of photoinduced electron-hole separation to improve energy transfer in catalytic reactions. Electron spin resonance (ESR) spectroscopy has been used to monitor the generation of electron/hole and reactive oxygen species (ROS) during nanomaterial-mediated photocatalysis. Using ESR coupled with spin trapping and spin labeling techniques, the underlying photocatalytic mechanism involved in the nanomaterial-mediated photocatalysis was investigated. In this review, we briefly introduced ESR principle and summarized recent advancements using ESR spectroscopy to characterize electron-hole separation and ROS production by different types of nanomaterials.

Tu-San-Qi (Gynura japonica): the culprit behind pyrrolizidine alkaloid-induced liver injury in China.

Herbs and dietary supplement-induced liver injury (HILI) is the leading cause of drug-induced liver injury in China. Among different hepatotoxic herbs, the pyrrolizidine alkaloid (PA)-producing herb Gynura japonica contributes significantly to HILI by inducing hepatic sinusoidal obstruction syndrome (HSOS), a liver disorder characterized by hepatomegaly, hyperbilirubinemia, and ascites. In China, G. japonica has been used as one of the plant species for Tu-San-Qi and is often misused with non-PA-producing Tu-San-Qi (Sedum aizoon) or even San-Qi (Panax notoginseng) for self-medication. It has been reported that over 50% of HSOS cases are caused by the intake of PA-producing G. japonica. In this review, we provide comprehensive information to distinguish these Tu-San-Qi-related herbal plant species in terms of plant/medicinal part morphologies, medicinal indications, and chemical profiles. Approximately 2156 Tu-San-Qi-associated HSOS cases reported in China from 1980 to 2019 are systematically reviewed in terms of their clinical manifestation, diagnostic workups, therapeutic interventions, and outcomes. In addition, based on the application of our developed mechanism-based biomarker of PA exposure, our clinical findings on the definitive diagnosis of 58 PA-producing Tu-San-Qi-induced HSOS patients are also elaborated. Therefore, this review article provides the first comprehensive report on 2214 PA-producing Tu-San-Qi (G. japonica)-induced HSOS cases in China, and the information presented will improve public awareness of the significant incidence of PA-producing Tu-San-Qi (G. japonica)-induced HSOS and facilitate future prevention and better clinical management of this severe HILI.

Comprehensive investigation and risk study on pyrrolizidine alkaloid contamination in Chinese retail honey.

Pyrrolizidine alkaloids (PAs) are common phytotoxins. We performed the first comprehensive investigation on PA contamination in Chinese honeys. LC-MS analysis revealed that 58% of 255 honey samples purchased from 17 regions across Mainland China and Taiwan contained PAs with total content ranging over 0.2-281.1 µg/kg. Monocrotaline (from Crotalaria spp), a PA never found in honey in other regions, together with echimidine (Echium plantagineum) and lycopsamine (from Senecio spp.), were three predominant PAs in PA-contaminated Chinese honeys. Further, PAs present in honeys were found to have geographically distinct pattern, indicating possible control of such contamination in future honey production. Moreover, we proposed a new risk estimation approach, which considered both content and toxic potency of individual PAs in honeys, and found that 12% of the PA-contaminated Chinese honeys tested might pose potential health risk. This study revealed a high prevalence and potential health risk of PA contamination in Chinese honeys.

1-Formyl-7-hydroxy-6,7-dihydro-5H-pyrrolizine (1-CHO-DHP)-Cysteine Conjugates: Metabolic Formation and Binding to Cellular DNA.

1-Formyl-7-hydroxy-6,7-dihydro-5H-pyrrolizine (1-CHO-DHP) is a potential proximate carcinogenic metabolite of pyrrolizidine alkaloids. In the present study, we determined that the reaction of 1-CHO-DHP with cysteine generated four identified products. By mass and 1H NMR spectral analyses, these products are cysteinyl-[2'-S-7]-1-CHO-DHP (P2), cysteinyl-[3'-N-7]-1-CHO-DHP (P3), 7-keto-DHP (P4), and 1-cysteinylimino-DHP (P5). These four compounds were also formed from the incubation of 1-CHO-DHP in HepG2 cells. Compounds P3 and P5 were interconvertible in acetonitrile and water. Incubation of P2 in HepG2 cells generated the four DHP-dG and -dA adducts that we propose to be potential common biomarkers of pyrrolizidine alkaloids exposure and pyrrolizidine alkaloids-induced liver tumor initiation. These four DHP-DNA adducts were also formed from the incubation of a mixture of P3 and P5 in HepG2 cells but not from the incubation with 7-keto-DHP. From the reaction of 1-CHO-DHP with glutathione, only trace amounts of the glutathione-1-CHO-DHP adduct were detected, with the structure unable to be characterized.

Effects of glutathione and cysteine on pyrrolizidine alkaloid-induced hepatotoxicity and DNA adduct formation in rat primary hepatocytes.

Pyrrolizidine alkaloids (PAs) are hepatotoxic, genotoxic, and carcinogenic phytochemicals. Upon metabolic activation, PAs produce dehydropyrrolizidine alkaloids (dehydro-PAs) as reactive primary pyrrolic metabolites. Dehydro-PAs are unstable, facilely hydrolyzed to (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP). Both dehydro-PAs and DHP are capable of binding to cellular DNA and proteins to form DHP-DNA and DHP-protein adducts leading to tumorigenicity and cytotoxicity. We recently determined that the reaction of dehydro-PAs with glutathione and cysteine generated 7-glutathione-DHP (7-GS-DHP) and 7-cysteine-DHP, respectively which can also bind to DNA to produce DHP-DNA adducts. In this study, we determined the effects of glutathione and cysteine on the induction of hepatocytotoxicity and the formation of DHP-DNA adducts in primary hepatocytes cultured with riddelliine and monocrotaline. We found that both glutathione and cysteine can drastically reduce hepatotoxicity while the levels of DHP-DNA adduct formation are slightly affected.

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.

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.

Quantitation of DNA reactive pyrrolic metabolites of senecionine - A carcinogenic pyrrolizidine alkaloid by LC/MS/MS analysis.

Pyrrolizidine alkaloids (PAs) are carcinogenic phytochemicals, inducing liver tumors in experimental rodents. We previously determined that (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP), 7-glutathione-DHP, 7-cysteine-DHP, 7-N-acetylcysteine-DHP, and 1-CHO-DHP are DNA reactive pyrrolic metabolites potentially associated with PA-induced liver tumor initiation. In this study, we developed an LC/MS/MS multiple reaction monitoring (MRM) mode method to identify and quantify these metabolites formed from the metabolism of senecionine, a carcinogenic PA, by mouse, rat, and human liver microsomes, and primary rat hepatocytes. Together with the chemically prepared standards of these metabolites, this represents an accurate and convenient LC/MS/MS analytical method for quantifying these five reactive pyrrolic metabolites in biological systems.

Influences of simulated gastrointestinal environment on physicochemical properties of gold nanoparticles and their implications on intestinal epithelial permeability.

Gold nanoparticles (Au NPs) hold great promise in food, industrial and biomedical applications due to their unique physicochemical properties. However, influences of the gastrointestinal tract (GIT), a likely route for Au NPs administration, on the physicochemical properties of Au NPs has been rarely evaluated. Here, we investigated the influence of GIT fluids on the physicochemical properties of Au NPs (5, 50, and 100 nm) and their implications on intestinal epithelial permeability in vitro. Au NPs aggregated in fasted gastric fluids and generated hydroxyl radicals in the presence of H2O2. Cell studies showed that GIT fluids incubation of Au NPs affected the cellular uptake of Au NPs but did not induce cytotoxicity or disturb the intestinal epithelial permeability.

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.