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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. Supplementary Information The online version contains supplementary material available at 10.1007/s00204-021-03129-6.

We recently established a genotoxic mechanism mediated by a set of (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5

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.

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.

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 reaction of 1-CHO-DHP...

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-5

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.

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. Keywords: Pyrrolizidine alkaloid, Senecionine, Pyrrolic metabolites, Carcinogenicity, LC/MS/MS analysis

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 PAsʼ 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.

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.

Combinations of semiconductor nanoparticles (NPs) with noble metal NPs enable an increase in the photoactivity of semiconductor NPs into the visible and near-infrared regions. The design ra...

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.

Traditional Chinese medicine (TCM) has long been used for treating illness in China and other Asian countries, and recently use d by the Western countries in several different ways, either for new drug development, or as functional foods and dietary supplement s. However, quality assurance and health adverse effects of the herbal plants have not been well studied. Pyrrolizidine alkaloids , a class of hepatotoxic and tumorigenic compounds, have been detected in herbal plants and dietary supplements. In this review, the source s of the pyrrolizidine alkaloid-containing Chinese herbal plants in China and the toxicity, genotoxicity, and tumorigenicity of these co mpounds are discussed. The metabolic pathways, particularly the activation pathways leading to genotoxicity, are discussed. Recent mec hanistic studies indicate that pyrrolizidine alkaloids induce tumors via a genotoxic mechanism mediated by 6,7-dihydro-7-hydroxy-1-hydro xymethyl-5H-pyrrolizine (DHP)-derived DNA adduct formation. This mechanism may be general to most carcinogenic pyrrolizidine alkaloids. Perspectives are included for suggestion of directions of future research.

1-Formyl-7-hydroxy-6,7-dihydro-5

Pyrrolizidine alkaloids (PAs) are one of the most significant groups of hepatotoxic phytotoxins. It is well-studied that metabolic activation of PAs generates reactive pyrrolic metabolites that rapidly bind to cellular proteins to form pyrrole-protein adducts leading to hepatotoxicity. Pyrrole-protein adducts all contain an identical core pyrrole moiety regardless of structures of the different PAs; however, the proteins forming pyrrole-protein adducts are largely unknown. The present study revealed that ATP synthase subunit beta (ATP5B), a critical subunit of mitochondrial ATP synthase, was a protein bound to the reactive pyrrolic metabolites forming pyrrole-ATP5B adduct. Using both anti-ATP5B antibody and our prepared anti-pyrrole-protein antibody, pyrrole-ATP5B adduct was identified in the liver of rats, hepatic sinusoidal endothelial cells, and HepaRG hepatocytes treated with retrorsine, a well-studied representative hepatotoxic PA. HepaRG cells were then used to further explore the consequence of pyrrole-ATP5B adduct formation. After treatment with retrorsine, significant amounts of pyrrole-ATP5B adduct were formed in HepaRG cells, resulting in remarkably reduced ATP synthase activity and intracellular ATP level. Subsequently, mitochondrial membrane potential and respiration were reduced, leading to mitochondria-mediated apoptotic cell death. Moreover, pre-treatment of HepaRG cells with a mitochondrial membrane permeability transition pore inhibitor significantly reduced retrorsine-induced toxicity, further revealing that mitochondrial dysfunction caused by pyrrole-ATP5B adduct formation significantly contributed to PA intoxication. Our findings for the first time identified ATP5B as a protein covalently bound to the reactive pyrrolic metabolites of PAs to form pyrrole-ATP5B adduct, which impairs mitochondrial function and significantly contributes to PA-induced hepatotoxicity.

Pyrrolizidine alkaloids (PAs) are phytotoxins identified in over 6000 plant species worldwide. Approximately 600 toxic PAs and PA N-oxides have been identified in about 3% flowering plants. PAs can cause toxicities in different organs particularly in the liver. The metabolic activation of PAs is catalyzed by hepatic cytochrome P450 and generates reactive pyrrolic metabolites that bind to cellular proteins to form pyrrole-protein adducts leading to PA-induced hepatotoxicity. The mechanisms that pyrrole-protein adducts induce toxicities have not been fully characterized. Methods for qualitative and quantitative detection of pyrrole-protein adducts have been developed and applied for the clinical diagnosis of PA exposure and PA-induced liver injury. This mini-review addresses the mechanisms of PA-induced hepatotoxicity mediated by pyrrole-protein adducts, the analytical methods for the detection of pyrrole-protein adducts, and the development of pyrrole-protein adducts as the mechanism-based biomarker of PA exposure and PA-induced hepatotoxicity. Keywords: Pyrrolizidine alkaloids, Pyrrole-protein adducts, Covalent binding, Target protein, Mechanism-based biomarker, Clinical diagnosis

Pyrrolizidine alkaloids (PAs) are natural toxins present in over 6000 plants. Humans are exposed to PAs through the intake of PA-containing plants and/or PA-contaminated foodstuffs, such as honey. PA contamination in honey has been extensively studied in many countries, but rarely investigated in China. The present study investigated PA contamination in retail honey purchased in four representative regions (Hong Kong, Macau, Beijing, Shanghai) in China. Among 120 honey samples tested, 58% of them contained PAs with the concentration range over 0.04–288.07 μg/kg. The prevalence and content of PAs in honeys purchased in Hong Kong and Macau were significantly higher than those detected in Beijing and Shanghai. Lycopsamine and echimidine were determined as predominant PAs in the contaminated honeys, and their potential botanic sources including plants of Echium and Senecio genera were identified by pollen analysis. Moreover, clivorine, which has not been reported previously in PA-contaminated honey, was found with a high prevalence (34%) in PA-contaminated honeys in China. Our study represented an integrated report on PA contamination in retail honey in China. The data provided information useful for the global assessment of human risk posed by consumption of PA-contaminated honey.

Engineered nanomaterials may adversely impact human health and environmental safety by nano-bio-eco interactions not fully understood. Their interaction with biotic and abiotic environments are varied and complicated, ranging from individual species to entire ecosystems. Their behavior, transport, fate, and toxicological profiles in these interactions, addressed in a pioneering study, are subsequently seldom reported. Biological, chemical, and physical dimension properties, the so-called multidimensional characterization, determine interactions. Intermediate species generated in the dynamic process of nanomaterial transformation increase the complexity of assessing nanotoxicity. We review recent progress in understanding these interactions, discuss the challenges of the study, and suggest future research directions.

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.

Although nanosized ingredients, including TiO2 nanoparticles (NPs), can be found in a wide range of consumer products, little is known about the effects these particles have on other active compounds in product matrices. These NPs can interact with reactive oxygen species (ROS), potentially disrupting or canceling the benefits expected from antioxidants. We used electron spin resonance spectrometry to assess changes in the antioxidant capacities of six dietary antioxidants (ascorbic acid, α-tocopherol, glutathione, cysteine, epicatechin, and epicatechin gallate) during exposure to P25 TiO2 and/or simulated sunlight. Specifically, we determined the ability of these antioxidants to scavenge 1-diphenyl-2-picryl-hydrazyl radical, superoxide radical, and hydroxyl radical. Exposure to simulated sunlight alone did not lead to noticeable changes in radical-scavenging abilities; however, in combination with P25 TiO2 NPs, the scavenging abilities of most antioxidants were weakened. We found glutathione to be the most resistant to treatment with sunlight and NPs among these six antioxidants.

There are 660 pyrrolizidine alkaloids (PAs) and PA N-oxides present in the plants, with approximately half being possible carcinogens. We previously reported that a set of four PA-derived DNA adducts is formed in the liver of rats administered a series of hepatocarcinogenic PAs and a PA N-oxide. Based on our findings, we hypothesized that this set of DNA adducts is a common biological biomarker of PA-induced liver tumor formation. In this study, we determined that rat liver microsomal metabolism of five hepatocarcinogenic PAs (lasiocarpine, retrorsine, riddelliine, monocrotaline, and heliotrine) and their corresponding PA N-oxides produced the same set of DNA adducts. Among these compounds, lasiocarpine N-oxide, retrorsine N-oxide, monocrotaline N-oxide, and heliotrine N-oxide are for first time shown to be able to produce these DNA adducts. These results further support the role of these DNA adducts as potential common biomarkers of PA-induced liver tumor initiation.

Many pyrrolizidine alkaloids (PAs) are hepatotoxic, genotoxic, and carcinogenic phytochemicals. Metabolism of PAs in vivo generates four (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-DNA adducts that have been proposed to be responsible for PA-induced liver tumor formation in rats. In this present study, we determined that the same set of DHP-DNA adducts was formed upon the incubation of 7-glutathione-DHP and 7-cysteine-DHP with cultured human hepatocarcinoma HepG2 cells. These results suggest that 7-glutathione-DHP and 7-cysteine-DHP are reactive metabolites of PAs that can bind to cellular DNA to form DHP-DNA adducts in HepG2 cells, and can potentially initiate liver tumor formation.

Pyrrolizidine alkaloids are among the most common poisonous plants affecting livestock, wildlife, and humans. Exposure of humans and livestock to toxic pyrrolizidine alkaloids through the intake of contaminated food and feed may result in poisoning, leading to devastating epidemics. During February 2014, 73 mixed breed female beef cows from the Galilee region of Israel were accidently fed pyrrolizidine alkaloid contaminated hay for 42 days, resulting in the sudden death of 24 cows over a period of 63 days. The remaining cows were slaughtered 2.5 months after the last ingestion of the contaminated hay. In this study, we report the histopathological analysis of the livers from five of the slaughtered cows and quantitation of pyrrolizidine alkaloid-derived DNA adducts from their livers and three livers of control cows fed with feed free of weeds producing pyrrolizidine alkaloids. Histopathological examination revealed that the five cows suffered from varying degrees of bile duct proliferation, fibrosis, and megalocytosis. Selected reaction monitoring HPLC-ES-MS/MS analysis indicated that (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-derived DNA adducts were formed in all five livers. The livers from the three control cows did not have any liver damage nor any indication of DHP-DNA adduct formed. These results confirm that the toxicity observed in these cattle was caused by pyrrolizidine alkaloid poisoning and that pyrrolizidine alkaloid-derived DNA adducts could still be detected and quantified in the livers of the chronically poisoned cows 2.5 months after their last exposure to the contaminated feed, suggesting that DHP-derived DNA adducts can serve as biomarkers for pyrrolizidine alkaloid exposure and poisoning.

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

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.

Combinations of semiconductor nanoparticles (NPs) with noble metal NPs enable an increase in the photoactivity of semiconductor NPs into the visible and near-infrared regions. The design rationale of the semiconductor-metal hybrid nanostructures for the optimization of charge carrier separation and reactive oxygen species (ROS) generation remains unclear. In this study, the interactions of Au nanorods (AuNRs) with TiO

Pyrrolizidine alkaloids (PAs) are phytochemicals present in more than 6000 plant species worldwide; about half of the PAs are hepatotoxic, genotoxic, and carcinogenic. Because of their wide exposure and carcinogenicity, the International Programme on Chemical Safety (IPCS) concluded that PAs are a threat to human health and safety. We recently determined that PA-induced liver tumor initiation is mediated by a set of four (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5 H-pyrrolizine (DHP)-DNA adducts and proposed that these DHP-DNA adducts are biomarkers of PA exposure and liver tumor initiation. To validate the generality of this metabolic activation pathway and DHP-DNA adducts as biomarkers, it is significant to identify reactive metabolites associated with this metabolic activation pathway. Segall et al. ( Segall et al. ( 1984 ) Drug Metab. Dispos. 12 , 68 - 71 ) previously reported that 1-formyl-7-hydroxy-6,7-dihydro-5 H-pyrrolizine (1-CHO-DHP) is generated from the metabolism of senecionine by mouse liver microsomes. In the present study, we examined the metabolism of seven hepatocarcinogenic PAs (senecionine, intermedine, retrorsine, riddelliine, DHR, heliotrine, and senkirkine) and one noncarcinogenic PA (platyphylline) by human, rat, and mouse liver microsomes. 1-CHO-DHP was identified as a common metabolite from the metabolism of these hepatotoxic PAs, but not from platyphylline. Incubation of 1-CHO-DHP with HepG2 and A549 cells produced the same set of DHP-DNA adducts, which were identified by both LC/MS MRM mode and selected ion monitoring analyses through comparison to synthetic standards. In the incubation medium of 1-CHO-DHP treated HepG2 cells, both DHP and 7-cysteine-DHP were formed, which were capable of binding to cellular DNA to produce DHP-DNA adducts. These results suggest that 1-CHO-DHP is a proximate DNA metabolite of genotoxic and carcinogenic PAs.

Pyrrolizidine alkaloids (PAs) are naturally occurring phytotoxins widely distributed in about 3% of flowering plants. The formation of PA-derived pyrrole-protein adducts is considered as a primary trigger initiating PA-induced hepatotoxicity. The present study aims to (i) further validate our previous established derivatization method using acidified ethanolic AgNO3 for the analysis of pyrrole-protein adducts and (ii) apply this method to characterize the binding tendency, dose-response, and elimination kinetics of pyrrole-protein adducts in blood samples. Two pyrrole-amino acid conjugates, (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5 H-pyrrolizine (DHP)-cysteine (7-cysteine-DHP) and 9-histidine-DHP, were synthesized and used to demonstrate that acidified ethanolic AgNO3 derivatization can cleave both S-linkage and N-linkage of pyrrole-protein adducts. Subsequently, using precolumn AgNO3 derivatization followed by ultra-high-pressure liquid chromatography/mass spectrometry analysis, we quantified pyrrole-protein adducts in monocrotaline-treated rat blood protein fractions, including hemoglobin (Hb), plasma, albumin, and plasma residual protein fractions, and found that the amount of pyrrole-Hb adducts was significantly higher than that in all plasma fractions. Moreover, elimination half-life of pyrrole-Hb adducts was also significantly longer than pyrrole-protein adducts in plasma fractions (12.08 vs 2.54-2.93 days). In addition, we also tested blood samples obtained from five PA-induced liver injury patients and found that the amount of pyrrole-protein adducts in blood cells was also remarkably higher than that in plasma. In conclusion, our findings for the first time confirmed that the AgNO3 derivatization method could be used to measure both S- and N-linked pyrrole-protein adducts and also suggested that pyrrole-Hb adducts with remarkably higher level and longer life span could be a better biomarker of PA exposure.