Riddelliine N-oxide is a phytochemical and mammalian metabolite with genotoxic activity that is comparable to the parent pyrrolizidine alkaloid riddelliine

Pyrrolizidine alkaloids (PAs) and their N-oxide derivatives are naturally-formed genotoxic phytochemicals that are widely distributed throughout the world. Although, the quantities of PAs and PA N-oxides in plants are nearly equal, the biological and genotoxic activities of PA N-oxides have not been studied extensively. PA N-oxides are major metabolites of PAs and are generally regarded as detoxification products. However, in this study, we report that rat liver microsomes converted riddelliine N-oxide to the genotoxic 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP) metabolite. Metabolism of riddelliine N-oxide by rat liver microsomes under hypoxic conditions (argon) generated predominantly the parent PA, riddelliine. The reduction of riddelliine N-oxide to riddelliine was diminished, when the metabolism of riddelliine N-oxide with rat liver microsomes was conducted aerobically. Rat liver microsomal incubations of riddelliine N-oxide in the presence of calf thymus DNA produced a set of DHP-derived DNA adducts as detected and quantified by 32P-postlabeling/HPLC. The same DHP-derived DNA adducts were also found in liver DNA of F344 rats fed riddelliine N-oxide or riddelliine. When rats received doses of 1.0 mg/kg riddelliine N-oxide for three consecutive days, the level of DNA adducts was 39.9 +/- 0.6 adducts/10(7) nucleotides, which was 2.6-fold less than that measured in rats treated with riddelliine at the same dose. We have previously shown that these DHP-derived DNA adducts are produced by chronic feeding of riddelliine and that the adduct levels correlated with liver tumor formation. Results presented in this paper indicate that riddelliine N-oxide, through its conversion to riddelliine, is also a potential genotoxic hepatocarcinogen.     

Formation of DHP-derived DNA adducts from metabolic activation of the prototype heliotridine-type pyrrolizidine alkaloid, heliotrine

Pyrrolizidine alkaloid-containing plants are widespread in the world and may be the most common poisonous plants affecting livestock, wildlife, and humans. Pyrrolizidine alkaloids require metabolism to exert their genotoxicity and tumorigenicity. Our mechanistic studies have determined that metabolism of the retronecine-type (riddelliine, retrorsine, and monocrotaline), heliotridine-type (lasiocarpine), and otonecine-type (clivorine) tumorigenic pyrrolizidine alkaloids in vivo and/or in vitro all generates a common set of 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-derived DNA adducts responsible for tumor induction. All the pyrrolizidine alkaloids studied previously are diesters with an ester linkage at the C7 and C9 positions of the necine base. In this study, we report that F344 rat liver microsomal metabolism of heliotrine, a tumorigenic monoester bearing a hydroxyl group at the C7 of the necine base, resulted in the formation of the dehydroheliotridine (DHH) metabolite. When incubations of heliotrine were carried out in the presence of calf thymus DNA, the same set of DHP-derived DNA adducts was formed. These results support that DHP-derived DNA adducts are potential common biomarkers of pyrrolizidine alkaloid exposure and tumorigenicity. For comparison, the dehydroretronecine (DHR)-derived DNA adducts formed from metabolism of riddleiine, retrorsine, monocrotaline, riddelleiine N-oxide, and retrorsine N-oxide were measured in parallel; the levels of DHP-derived DNA adduct formation were in the order: riddelliine approximately retrorsine>monocrotaline>retrorsine N-oxide>or=riddelliine N-oxide>heliotrine.     

Protection of epidermal growth factor against clivorine‐induced mitochondrial‐mediated apoptosis in hepatocytes

Pyrrolizidine alkaloids (PAs) are well‐known natural hepatotoxins. In this study, we investigated the protection of epidermal growth factor (EGF) against the hepatotoxicity of clivorine, which is an otonecine‐type PA from traditional Chinese medicine Ligularia hodgsonii Hook. Cell viability assay and cell morphology observation showed that EGF (1 ng/mL) reversed clivorine‐induced cytotoxicity on human normal liver L‐02 cells. EGF (1 ng/mL) also inhibited clivorine‐induced DNA fragmentation and caspase‐3 cleavage. Our previous study has showed that antiapoptotic Bcl‐xL degradation and mitochondrial‐mediated apoptosis was involved in clivorine‐induced hepatotoxicity. In this study, we found that EGF (1 ng/mL) inhibited clivorine‐induced antiapoptotic Bcl‐xL protein decrease, caspase‐9 activation, and release of cytosolic cytochrome C. We further investigated the effects of vascular epidermal growth factor, basic fibroblast growth factor, and insulin‐like growth factor‐1 on clivorine‐induced cytotoxicity, and there is no significant protection observed. Our results suggest that EGF exerts its protective effects against clivorine‐induced hepatotoxicity probably by modulating mitochondrial‐mediated apoptotic signal pathway. © 2009 Wiley Periodicals, Inc. Environ Toxicol, 2010.     

Human liver microsomal metabolism and DNA adduct formation of the tumorigenic pyrrolizidine alkaloid,riddelliine

Riddelliine, a widespread naturally occurring genotoxic pyrrolizidine alkaloid, induced liver tumors in rats and mice in an NTP 2-year carcinogenicity bioassay. We have determined that riddelliine induces liver tumors in rats through a genotoxic mechanism involving the formation of (+/-)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP), which reacts with DNA to form a set of eight DNA adducts. To determine the relevance to humans of the results obtained in experimental animals, the metabolism of riddelliine was conducted using human liver microsomes. As with rat liver microsomes, DHP and riddelliine N-oxide were major metabolites in incubations conducted with human liver microsomes. The levels of DHP and riddelliine N-oxide were 0.20-0.62 and 0.03-0.15 nmol/min/mg protein, respectively, which are comparable to those obtained from rat liver microsomal metabolism. When metabolism was conducted in the presence of calf thymus DNA, the same set of eight DHP-derived DNA adducts was formed. Both the metabolism pattern and DNA adduct profile were very similar to those obtained from rat liver microsomes. When metabolism was conducted in the presence of the P450 3A4 enzyme inhibitor triacetyleandomycin, the formation of DHP and riddelliine N-oxide was reduced 84 and 92%, respectively. For DHP formation, the Km values were determined to be 0.37 +/- 0.05 and 0.66 +/- 0.08 mM from female rats and female humans; the Vmax values from female rat and human liver microsomal metabolism were 0.48 +/- 0.03 and 1.70 +/- 0.09 nmol/min/mg protein, respectively. These results strongly indicate the mechanistic data on liver tumor induction obtained for riddelliine in laboratory rodents is highly relevant to humans.     

Identification of DNA adducts derived from riddelliine,a carcinogenic pyrrolizidine alkaloid

Riddelliine is a naturally occurring carcinogenic pyrrolizidine alkaloid that produces liver tumors in experimental animals. Riddelliine requires metabolic activation to dehydroriddelliine and 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP) to exert its toxicity. Previously, (32)P-postlabeling HPLC was used to detect a set of eight DHP-derived adduct peaks from DNA modified both in vitro and in vivo. Among these DHP-derived DNA adducts, two were identified as epimers of DHP-2'-deoxyguanosine 3'-monophosphate. In this study, the remaining adducts have been characterized as DHP-modified dinucleotides. A series of dinucleotides, TpGp, ApGp, TpCp, ApCp, TpAp, ApAp, TpTp, and ApTp, were obtained by enzymatic digestion of calf thymus DNA with micrococcal nuclease (MN) and spleen phosphodiesterase (SPD) followed by HPLC separation and structural identification by negative ion electrospray tandem mass spectrometry (ES/MS/MS). Incubation of individual dinucleotides with DHP produced DHP-modified dinucleotide adducts that were also characterized using LC-ES/MS/MS. A parallel analysis of the isolated DHP-modified dinucleotides using (32)P-postlabeling recapitulated the series of unidentified adduct peaks that we previously reported from DHP-modified calf thymus DNA in vitro and rat liver DNA in vivo. Intact calf thymus DNA was also reacted with DHP and then digested by MN/SPD under the same conditions. The adduct profile obtained from LC-ES/MS/MS analysis was similar to that observed from the isolated dinucleotides. Structural analysis using LC-ES/MS/MS showed that DHP bound covalently to both 3'- and 5'-guanine, -adenine, and -thymine bases (but not cytosine) of dinucleotides to produce two or more isomers of each DHP-dinucleotide adduct. By comparing adduct formation at dissimilar bases within individual dinucleotides, the relative reactivity of DHP with individual bases was determined to be guanine > adenine approximately thymine. Identification of the entire set of DHP-derived DNA adducts further validates the conclusion that riddelliine is a genotoxic carcinogen and enhances the applicability of these biomarkers for assessing carcinogenic risks from exposure to pyrrolizidine alkaloids.     

Human liver microsomal reduction of pyrrolizidine alkaloid N-oxides to form the corresponding carcinogenic parent alkaloid

Retronecine-based pyrrolizidine alkaloids, such as riddelliine, retrorsine, and monocrotaline, are toxic to domestic livestock and carcinogenic to laboratory rodents. Previous in vitro metabolism studies showed that (+/-)6,7-dihydro-7-hydroxy-1-(hydroxymethyl)-5H-pyrrolizine (DHP) and pyrrolizidine alkaloid N-oxides were the major metabolites of these compounds. DHP is the reactive metabolite of pyrrolizidine alkaloids and pyrrolizidine alkaloid N-oxides are generally regarded as detoxification products. However, a previous study of rat liver microsomal metabolism of riddelliine N-oxide demonstrated that DHP and its parent compound, riddelliine, were generated as the major metabolites of riddelliine N-oxide. In this study the metabolic activation of the three retronecine-based pyrrolizidine alkaloid N-oxides by human liver microsomes is investigated under oxidative and hypoxic conditions. Results shows that both the DHP and the corresponding parent pyrrolizidine alkaloids are the major metabolites of the human liver microsomal metabolism of pyrrolizidine alkaloid N-oxides. Under oxidative conditions, reduction of the N-oxide to pyrrolizidine alkaloid is inhibited and while under hypoxic conditions, DHP formation is dramatically decreased. The oxidative and reductive products generated from the metabolism of pyrrolizidine alkaloid N-oxides are substrate-, enzyme- and time-dependent. In the presence of troleandomycin, a microsomal CYP3A inhibitor, DHP formation is inhibited by more than 70%, while the N-oxide reduction was not affected. The level of microsomal enzyme activity in human liver is comparable with rats. The rate of in vitro metabolism by either human and rat liver microsomes follows the order of riddelliine > or = retrorsine > monocrotaline, and DHP-derived DNA adducts are detected and quantified by 32P-postlabeling/HPLC analysis. Similar DHP-derived DNA adducts are found in liver DNA of F344 rats gavaged with the pyrrolizidine alkaloid N-oxides (1.0 mg/kg). The levels of in vivo DHP-DNA adduct formation is correlated with the level of in vitro DHP formation. Our results indicate that pyrrolizidine alkaloid N-oxides may be hepatocarcinogenic to rats through a genotoxic mechanism via the conversion of the N-oxides to their corresponding parent pyrrolizidine alkaloids, and these results may be relevant to humans.     

Pyrrole-protein adducts – A biomarker of pyrrolizidine alkaloid-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.     

人肝CYP3A参与了山冈橐吾碱的代谢及其肝毒性代谢物的形成(英文)

目的　在体外研究山冈橐吾碱在人肝微粒体内的代谢及参与其代谢的主要的CYP4 5 0酶 ,探讨其代谢致毒机理。方法　采用人肝微粒体研究山冈橐吾碱的主要代谢方式和代谢物。在体外运用CYP4 5 0酶的选择性抑制剂和cDNA表达的人肝CYP4 5 0酶 ,探讨其对山冈橐吾碱的代谢及肝毒性的吡咯代谢物形成的影响及参与山冈橐吾碱代谢的主要的CYP4 5 0酶。结果　山冈橐吾碱在人肝微粒体内的主要代谢物为肝毒性的吡咯代谢物 :去氢倒千里光裂碱 ,7 谷胱甘肽基 去氢倒千里光裂碱 ,7,9 二谷胱甘肽基去氢倒千里光裂碱和山冈囊吾酸。CYP4 5 0特异性抑制剂α 萘黄酮 (抑制CYP1A2 )、黄胺苯吡唑 (抑制CYP2C)、奎尼丁 (抑制CYP2D6 )和二乙基二硫代氨基甲酸钠 (抑制CYP2E1)对山冈橐吾碱的代谢无明显的影响。但CYP3A的特异性抑制剂酮康唑和三乙酰竹桃霉素可以显著地抑制山冈橐吾碱的代谢及其吡咯代谢物和结合型吡咯物的形成。此外 ,在cDNA表达的人肝CYP3A4的温孵液中 ,山冈橐吾碱被代谢成相应的吡咯代谢物 ,而山冈橐吾碱在cDNA表达的人肝CYP1A2、CYP2C9、CYP2D6和CYP2E1温孵液中无代谢。结论　山冈橐吾碱在人肝微粒体内的主要代谢方式是形成肝毒性吡咯代谢物 ,CYP3A作为主要的CYP4 5 0酶参与了山冈橐吾碱的代谢及其肝毒性吡咯代谢?     

Characterization of rat liver microsomal metabolites of clivorine, an hepatotoxic otonecine-type pyrrolizidine alkaloid.

The metabolism of the hepatotoxic otonecine-type pyrrolizidine alkaloid (PA), clivorine, was investigated using rat liver microsomes. The metabolites dehydroretronecine (DHR), 7-glutathionyldehydroretronecine (7-GSH-DHR), 7, 9-diglutathionyldehydroretronecine (7,9-diGSH-DHR), and clivoric acid were identified using chromatographic and mass spectrometric analyses. NMR characterizations were also performed on the isolated clivoric acid and the synthetic 7-GSH-DHR and 7,9-diGSH-DHR. The results indicated that the two glutathione (GSH) conjugates were formed by reaction of the unstable toxic pyrrolic ester with GSH added in the microsomal incubation system, whereas DHR was generated from hydrolysis of the unstable pyrrolic ester, and that clivoric acid was produced from all these further conversions of the unstable pyrrolic ester. Furthermore, tissue-bound pyrroles were also determined to be present after microsomal incubation of clivorine. Clivoric acid has not been previously identified, and DHR and 7, 9-diGSH-DHR were found, for the first time, as metabolites of an otonecine-type PA, while 7-GSH-DHR was previously reported by us to be a microsomal metabolite of clivorine. The in vitro metabolic pathway of clivorine was delineated to be the initial formation of the unstable pyrrolic ester, which then may undergo hydrolysis, GSH conjugations, or covalent binding with hepatic tissues that may lead to hepatotoxicity. The present definitive identification of four pyrrolic ester-related metabolites of clivorine and indirect determination of bound pyrroles provide the strongest evidence to date to support the hypothesis that the formation of an unstable pyrrolic ester plays a key role in otonecine-type PA-induced hepatotoxicity.     

7-N-Acetylcysteine-pyrrole conjugate—A potent DNA reactive metabolite of pyrrolizidine alkaloids

Plants containing pyrrolizidine alkaloids (PAs) are widespread throughout the world and are the most common poisonous plants affecting livestock, wildlife, and humans. PAs require metabolic activation to form reactive dehydropyrrolizidine alkaloids (dehydro-PAs) that are capable of alkylating cellular DNA and proteins, form (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-DNA and DHP-protein adducts, and lead to cytotoxicity, genotoxicity, and tumorigenicity. In this study, we determined that the metabolism of riddelliine and monocrotaline by human and rat liver microsomes in the presence of N-acetylcysteine both produced 7-N-acetylcysteine-DHP (7-NAC-DHP) and DHP. Reactions of 7-NAC-DHP with 2′-deoxyguanosine (dG), 2′-deoxyadenosine (dA), and calf thymus DNA in aqueous solution followed by enzymatic hydrolysis yielded DHP-dG and/or DHP-dA adducts. These results indicate that 7-NAC-DHP is a reactive metabolite that can lead to DNA adduct formation.     

Gender differences in microsomal metabolic activation of hepatotoxic clivorine in rat

The gender differences in the in vitro microsomal metabolic activation of hepatotoxic clivorine, a representative naturally occurring hepatotoxic otonecine type pyrrolizidine alkaloid, in Sprague-Dawley rats and their relation to the gender differences in susceptibility to clivorine intoxication were reported in the present study. Clivorine-induced liver damage in the male rat via metabolic activation to form the reactive pyrrolic ester followed by covalent binding to liver tissue constituents has been reported previously by our research group. The present study demonstrated, for the first time, that cytochromes p450 3A1 and 3A2, which are constitutively expressed in the male rat, might play a significant role in the metabolic activation of clivorine in the rat. Thus, in the male rat, the metabolic activation by liver microsomes to form the reactive pyrrolic ester was found as the only direct metabolic pathway of clivorine followed by subsequent formation of the toxic tissue-bound pyrroles leading to hepatotoxicity. In the case of the female rat, a less significant metabolic activation was observed, whereas the formations of two novel nonpyrrolic metabolites were determined as the predominant biotransformations. None of the four cDNA-expressed rat enzymes (cytochrome p450 2C12, 2E1, 3A1, 3A2) tested could catalyze the formation of these two new metabolites. Furthermore, the female rat (LD(50) = 114 +/- 9 mg/kg, i.p.) was found to be significantly less susceptible to clivorine intoxication than the male rat (LD(50) = 91 +/- 3 mg/kg, i.p.). Therefore, the results suggested that a significantly lower metabolic activation due to the lack of cytochrome p450 3A1 and p450 3A2 activities mainly accounted for the smaller susceptibility of the female rat to clivorine intoxication.     

Species differences in the in vitro metabolic activation of the hepatotoxic pyrrolizidine alkaloid clivorine

Clivorine is a representative naturally occurring hepatotoxic otonecine-type pyrrolizidine alkaloid. Our previous study has demonstrated that clivorine induces liver damage via metabolic activation to form the reactive pyrrolic ester followed by covalent binding to liver tissue constituents. The present study investigated species differences in the in vitro metabolic activation of clivorine in the male rat and guinea pig of both sexes. In the male rat, the activation of clivorine to form the reactive pyrrolic ester was found as the only metabolic pathway. Moreover, the toxic tissue-bound pyrroles and four isolatable metabolites identified, namely DHR, 7-GSH-DHR, 7,9-diGSH-DHR, and clivoric acid, were all generated from further metabolism of this reactive intermediate. In the case of both sexes of guinea pig, the same activation was observed as the minor biotransformation, while an additional metabolic pathway, a direct hydrolysis of clivorine to form novel clivopic acid was identified as the predominant detoxification pathway. Furthermore, the formation rates for the toxic tissue-bound pyrroles and less toxic DHR were significantly slower and higher, respectively, compared with those in the male rat. In addition, the formation of the reactive pyrrolic ester was mediated by the CYP3A subfamily in both animals, while carboxylesterases might be responsible for the detoxification hydrolysis in guinea pig. The results suggest that the higher metabolic rates for detoxification hydrolyses and the lower formation rate for the toxic tissue-bound pyrroles play the key roles in guinea pig resistance to clivorine intoxication. Therefore, the male rat and guinea pig should be the suitable animal models for further studies of bioactivation and deactivation of otonecine-type PA, respectively.     

Species differences in the hepatic microsomal enzyme metabolism of the pyrrolizidine alkaloids

Species differences in pyrrolic metabolites and senecionine (SN) N-oxide formation among eight animal species (sheep, cattle, gerbils, rabbits, hamsters, Japanese quail, chickens, rats) varying in susceptibility to pyrrolizidine alkaloid (PA) intoxication were measured in vitro by hepatic microsomal incubations. The results suggested that there is not a strong correlation between the production of pyrrolic metabolites and susceptibility of animals to PA toxicity. The rate of PA activation in hamsters, a resistant species, measured by formation of (±)6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP) far exceeded the rate of SN N-oxide formation (detoxification) (DHP/N-oxide=2.29). In contrast, SN N-oxide was the major metabolite in sheep, another resistant species, with much lower production of DHP (DHP/N-oxide=0.26). The roles of cytochrome P450s and flavin-containing monooxygenases (FMO) in bioactivation and detoxification of pyrrolizidine alkaloids (PA) were studied in vitro using sheep and hamster hepatic microsomes. Chemical and immunochemical inhibition data suggested that the conversion of SN to DHP is catalyzed mainly by cytochrome P450s (68–82%), whereas the formation of SN N-oxide is carried out largely by FMO (55–71%). There also appeared to be a high rate of glutathione–DHP conjugation in hamster (63%) and sheep (79%) liver microsomal incubation mixtures. Therefore, low rates of pyrrole metabolite production coupled with glutathione conjugation in sheep may explain the resistance of sheep to SN, whereas the high rate of GSH-DHP conjugation may be one of the factors contributing to the resistance of hamsters to intoxication by this PA.     

Comparison of metabolism-mediated effects of pyrrolizidine alkaloids in a HepG2/C3A cell-S9 co-incubation system and quantification of their glutathione conjugates

Toxicity of pyrrolizidine alkaloids (PAs) largely depends on their metabolic activation by hepatic enzymes, including cytochrome P450s, to become chemically reactive pyrrolic derivatives. These then spontaneously release the esterifying acids to generate carbonium ions that form covalent adducts with cellular nucleophiles to exhibit toxicity. In our investigation, metabolism-mediated toxicity of monocrotaline, retrorsine, lycopsamine, echimidine (retronecine-type PAs), heliotrine (a heliotridine-type PA) and senkirkine (an otonecine-type PA) was studied using an in vitro co-incubation assay. Human hepatocarcinoma (HepG2/C3A) cells were incubated with PAs in the presence and absence of rat liver S9 fraction and the toxicity was assessed as lowered mitochondrial activity. Bioactivation potential was measured by incubating PAs with rat liver S9 fraction, NADPH and GSH in a cell free system. Pyrrolic metabolites generated were entrapped as glutathione conjugates (7-GSH-DHP and 7,9-di-GSH-DHP) which were quantified using LC-MS-MS analysis. Our results indicated that PAs were metabolized by rat liver S9 fraction into reactive pyrrolic derivatives which were toxic to HepG2/C3A cells. This approach can be used to determine and compare bioactivation potential and metabolism-mediated toxicity of various PAs.     

Genotoxicity of pyrrolizidine alkaloids

Pyrrolizidine alkaloids (PAs) are common constituents of many plant species around the world. PA‐containing plants are probably the most common poisonous plants affecting livestock and wildlife. They can inflict harm to humans through contaminated food sources, herbal medicines and dietary supplements. Half of the identified PAs are genotoxic and many of them are tumorigenic. The mutagenicity of PAs has been extensively studied in different biological systems. Upon metabolic activation, PAs produce DNA adducts, DNA cross‐linking, DNA breaks, sister chromatid exchange, micronuclei, chromosomal aberrations, gene mutations and chromosome mutations in vivo and in vitro. PAs induced mutations in the cII gene of rat liver and in the p53 and K‐ras genes of mouse liver tumors. It has been suggested that all PAs produce a set of (±)‐6,7‐dihydro‐7‐hydroxy‐1‐hydroxymethyl‐5H‐pyrrolizine‐derived DNA adducts and similar types of gene mutations. The signature types of mutations are G : C → T : A transversion and tandem base substitutions. Overall, PAs are mutagenic in vivo and in vitro and their mutagenicity appears to be responsible for the carcinogenesis of PAs. Published in 2010 by John Wiley & Sons, Ltd.     

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.     

Photoirradiation of dehydropyrrolizidine alkaloids--formation of reactive oxygen species and induction of lipid peroxidation

Pyrrolizidine alkaloid (PA)-containing plants are widespread in the world and are probably the most common poisonous plants affecting livestock, wildlife, and human. PAs require metabolic activation to generate pyrrolic metabolites (dehydro-PAs) that bind cellular protein and DNA, leading to hepatotoxicity and genotoxicity, including tumorigenicity. In this study we report that UVA photoirradiation of a series of dehydro-PAs, e.g., dehydromonocrotaline, dehydroriddelliine, dehydroretrorsine, dehydrosenecionine, dehydroseneciphylline, dehydrolasiocarpine, dehydroheliotrine, and dehydroretronecine (DHR) at 0-70 J/cm² in the presence of a lipid, methyl linoleate, resulted in lipid peroxidation in a light dose-responsive manner. When irradiated in the presence of sodium azide, the level of lipid peroxidation decreased; lipid peroxidation was enhanced when methanol was replaced by deuterated methanol. These results suggest that singlet oxygen is a photo-induced product. When irradiated in the presence of superoxide dismutase, the level of lipid peroxidation decreased, indicating that lipid peroxidation is also mediated by superoxide. Electron spin resonance (ESR) spin trapping studies confirmed that both singlet oxygen and superoxide anion radical were formed during photoirradiation. These results indicate that UVA photoirradiation of dehydro-PAs generates reactive oxygen species (ROS) that mediated the initiation of lipid peroxidation. UVA irradiation of the parent PAs and other PA metabolites, including PA N-oxides, under similar experimental conditions did not produce lipid peroxidation. It is known that PAs induce skin cancer and are secondary (hepatogenous) photosensitization agents. Our results suggest that dehydro-PAs are the active metabolites responsible for skin cancer formation and PA-induced secondary photosensitization.     

山冈橐吾碱在雌性大鼠肝微粒体内的代谢(英文)

研究了山冈橐吾碱 (clivorine)在雌性大鼠肝微粒体内的代谢 .山冈橐吾碱在雌性大鼠肝微粒体内的主要代谢物为两个非吡咯代谢物M1和M2 .与雄性大鼠不同 ,生成肝毒性的吡咯代谢物为其次要的代谢途径 .文献报道山冈橐吾碱在雄性大鼠肝微粒体内的主要代谢方式是形成相应的吡咯代谢物 .这提示山冈橐吾碱在雌雄大鼠肝微粒体内的主要代谢方式不同 .CYP4 5 0特异性抑制剂黄胺苯吡唑(CYP2C) ,毛果芸香碱 (CYP2A1) ,二乙基二硫代氨基甲酸钠 (CYP2E1)和酮康唑 (CYP3A)对M1和M2的形成无明显的影响 .黄素单氧化酶的特异性抑制剂甲巯咪唑可以显著地抑制M2 的形成 ,但对M1的形成无明显的抑制作用 ,且M1在肝微粒体中的形成为NADPH非依赖性 ,上述结果提示参与M1和M2代谢的酶分别为肝微粒体中的水解酶和黄素单氧化酶 .另一方面 ,毛果芸香碱 ,黄胺苯吡唑和二乙基二硫代氨基甲酸钠对山冈橐吾碱的吡咯代谢物的形成无明显的影响 ,而CYP3A的特异性抑制剂酮康唑可以显著地抑制吡咯代谢物的生成 ,且山冈橐吾碱在重组的大鼠肝CYP2C12 ,CYP2E1温孵液中无代谢 ,而在重组的大鼠肝CYP3A1和CYP3A2的温孵液中山冈橐吾碱被代谢成相应的吡咯代谢物 .这提示CYP3A作为主要的CYP4 5 0酶参与了山冈橐吾碱的肝毒性吡咯代谢物的形成 .山冈橐吾碱?     

Pyrrolizidine alkaloid-derived DNA adducts are common toxicological biomarkers of pyrrolizidine alkaloid N-oxides

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.     

Involvement of Bcl-xL degradation and mitochondrial-mediated apoptotic pathway in pyrrolizidine alkaloids-induced apoptosis in hepatocytes

Pyrrolizidine alkaloids (PAs) are natural hepatotoxins with worldwide distribution in more than 6000 high plants including medicinal herbs or teas. The aim of this study is to investigate the signal pathway involved in PAs-induced hepatotoxicity. Our results showed that clivorine, isolated from Ligularia hodgsonii Hook, decreased cell viability and induced apoptosis in L-02 cells and mouse hepatocytes. Western-blot results showed that clivorine induced caspase-3/-9 activation, mitochondrial release of cytochrome c and decreased anti-apoptotic Bcl-xL in a time (8-48 h)- and concentration (1-100 μM)-dependent manner. Furthermore, inhibitors of pan-caspase, caspase-3 and caspase-9 significantly inhibited clivorine-induced apoptosis and rescued clivorine-decreased cell viability. Polyubiquitination of Bcl-xL was detected after incubation with 100 μM clivorine for 40 h in the presence of proteasome specific inhibitor MG132, indicating possible degradation of Bcl-xL protein. Furthermore, pretreatment with MG132 or calpain inhibitor I for 2 h significantly enhanced clivorine-decreased Bcl-xL level and cell viability. All the other tested PAs such as senecionine, isoline and monocrotaline decreased mouse hepatocytes viability in a concentration-dependent manner. Clivorine (10 μM) induced caspase-3 activation and decreased Bcl-xL was also confirmed in mouse hepatocytes. Meanwhile, another PA senecionine isolated from Senecio vulgaris L also induced apoptosis, caspase-3 activation and decreased Bcl-xL in mouse hepatocytes. In conclusion, our results suggest that PAs may share the same hepatotoxic signal pathway, which involves degradation of Bcl-xL protein and thus leading to the activation of mitochondrial-mediated apoptotic pathway.