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.

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.

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.

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.

1-Formyl-7-hydroxy-6,7-dihydro-5 H-pyrrolizine (1-CHO-DHP): A Potential Proximate Carcinogenic Metabolite of Pyrrolizidine Alkaloids.

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.

Separation of charge carriers and generation of reactive oxygen species by TiO2 nanoparticles mixed with differently-coated gold nanorods under light irradiation.

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 TiO2 NPs were modulated by controlling their surface charges. Positively charged AuNRs formed aggregates with the negatively charged TiO2 NPs (AuNR@CTAB/TiO2) upon mixing, suggesting that Schottky junctions may exist between Au and TiO2. In contrast, negatively charged AuNRs (AuNR@PSS) remained spatially separated from the TiO2 NPs in the mixed suspension (AuNR@PSS/TiO2), owing to electrostatic repulsion. We used electron spin resonance (ESR) spectroscopy to detect the separation of charged carriers and ROS generation in these two mixtures under simulated sunlight irradiation. We also explored the role of dissolved oxygen in charge carrier separation and ROS generation by continuously introducing oxygen into the AuNR@CTAB/TiO2 suspension under simulated sunlight irradiation. Moreover, the generation of ROS by the AuNR@CTAB/TiO2 and AuNR@PSS/TiO2 mixtures were also examined under 808 nm laser irradiation. Our results show that the photogenerated electrons of excited semiconductor NPs are readily transferred to noble metal NPs simply by collisions, but the transfer of photogenerated hot electrons from excited AuNRs to TiO2 NPs is more stringent and requires the formation of Schottky junctions. In addition, the introduction of oxygen is an efficient way to enhance the photocatalytic activity of semiconductor NPs/noble metal NPs system combinations.

Pyrrole-Hemoglobin Adducts, a More Feasible Potential Biomarker of Pyrrolizidine Alkaloid Exposure.

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.

Primary and secondary pyrrolic metabolites of pyrrolizidine alkaloids form DNA adducts in human A549 cells.

Humans and animals can be exposed to carcinogenic pyrrolizidine alkaloids (PAs) through consumption of plants commonly found in many parts of the world. Although the liver is the primary target organ for carcinogenic PAs, they have also induced lung tumors in rodents. Hepatic cytochrome P450 activity converts PAs into dehydro-PAs that can be hydrolyzed to dehydropyrrolizidine (DHP); these reactive pyrrolic metabolites can produce four characteristic DNA adducts associated with PA-induced liver tumor initiation in laboratory animals. We reported recently that these four DNA adducts are also formed when 7-glutathione-DHP (7-GS-DHP) or 7-cysteine-DHP is incubated with calf thymus DNA. Here we showed that the four characteristic DNA adducts were formed when human A549 brochoalveolar carcinoma cells were treated with three dehydro-PAs (dehydroriddelliine, dehydromonocrotaline, or dehydroretronecine) or with 7-GS-DHP or 7-cysteine-DHP. For comparison, two parent PAs (riddelliine and monocrotaline) and 7,9-di-glutathionine-DHP were studied. No DHP-DNA adducts were detected with these incubations, confirming that A549 lung carcinoma cells do not express cytochrome P450 enzymes required for metabolic activation of PAs. Our results show that primary and secondary pyrrolic metabolites of carcinogenic PAs produce characteristic DHP-containing DNA adducts in A549 lung cancer cells, suggesting that they are DNA reactive metabolites.

The role of formation of pyrrole-ATP synthase subunit beta adduct in pyrrolizidine alkaloid-induced hepatotoxicity.

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.