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.     

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.     

Metabolism of clivorine, an otonecine-type hepa- totoxic pyrrolizidine alkaloid

AIM: To investigate pyrrolic metabolites of clivorine andinvolvement of such metabolites in inducing hepatotoxicity.METHODS: Microsomes were prepared from Sprague-Dawleyrats. Clivorine, an otonecine--type pyrrolizidine alkaloid (PA),was incubated with rat microsome in either presence or absence ofglutathione (GSH ). Samples obtained after incubation werecentrifuged, and aliquots of the supernatant were directly analyzedby HPLC--MS and quantified by HPLC--UV method with coupled-     

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.     

Metabolic formation of DHP-derived DNA adducts from a representative otonecine type pyrrolizidine alkaloid clivorine and the extract of Ligularia hodgsonnii hook

Plants that contain pyrrolizidine alkaloids (PAs) are widely distributed, and PAs have been shown to be genotoxic and tumorigenic in experimental animals. Our recent mechanistic studies indicated that riddelliine, a tumorigenic retronecine type PA, induced tumors via a genotoxic mechanism mediated by the formation of a set of eight 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-derived DNA adducts. However, it is not known if this mechanism is general to PAs of other types. In this study, we report that the metabolism of clivorine, a tumorigenic otonecine type PA, by F344 rat liver microsomes results in DHP formation. When incubations were conducted with clivorine in the presence of calf thymus DNA, eight DHP-derived DNA adducts were formed. The Ligularia hodgsonnii Hook plant, an antitussive traditional Chinese medicine, was found to contain otonecine type PAs with clivorine being predominant. DHP and DHP-derived DNA adducts were also obtained when microsomal incubations were conducted with extracts of L. hodgsonnii Hook. This is the first report that DHP-derived DNA adducts are formed from the metabolic activation of otonecine type PA and that these DHP-derived DNA adducts are potential biomarkers of PA exposure and PA-induced tumorigenicity. These results also provide evidence that the principal metabolic activation pathway of clivorine leading to liver genotoxicity and tumorigenicity is (i) formation of the corresponding dehydropyrrolizidine (pyrrolic) derivative through oxidative N-demethylation of the necine base followed by ring closure and dehydration and (ii) binding of the pyrrolic metabolite to DNA leading to the DNA adduct formation and tumor initiation.     

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.     

Genotoxicity of the pyrrolizidine alkaloid jacobine in rats

Jacobine (JAC) is a pyrolizidine alkaloid (PA) exhibiting adverse hepatic effects similar to those induced by another PA, monocrotaline (MCT). The in vitro reaction kinetics of JAC, however, have been reported to differ quantitively from those of MCT. We report results of experiments to detect and characterize hepatic DNA damage resulting from in vivo administration of JAC (5-60 mg/kg i.p.) to male Sprague-Dawley rats. Hepatic nuclei were isolated and served as the source of DNA in these experiments. Alkaline elution was employed to characterize the type(s) of DNA damage induced. At 4 h post administration, JAC induced significant dose-dependent DNA-DNA interstrand cross-linking over the entire range of doses. Significant DNA-protein cross-linking was also induced by doses of 15-60 mg/kg. No DNA single-strand breaks were detected. Previous studies in this laboratory have shown MCT to induce these same types of lesions. Results from these experiments demonstrate that despite a reported difference in vitro reaction kinetics, these compounds induce a similar spectrum of DNA damage in the target organ of a susceptible species, where the adverse effects induced are also similar. such similarities are consistent with the involvement of DNA damage in the adverse hepatic effects of PAs.     

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?     

Hepatotoxicity and tumorigenicity induced by metabolic activation of pyrrolizidine alkaloids in herbs

In the recent decades, the use of herbal products has been rapidly growing in the Western countries. While their use in many cases causes adverse effects, to date, safety issues of herbal products have not been adequately addressed. It is rarely determined whether the non-purported bioactive constituents in the herbs and the metabolites of the bioactive components can lead to adverse effects. In this review, we discuss, using pyrrolizidine alkaloids (PAs) as an example, the hepatotoxicity and tumorigenicity induced by metabolic activation of herbal components and by herb-herb and herb-drug interactions with other herbal ingredients and synthetic drugs. PAs are constitutively produced by plants as the secondary metabolites. There are more than 600 PAs and PA N-oxides identified in over 6000 plants, and more than half of them exhibit hepatotoxicity. Toxic PA-containing plants grow in many geographical regions worldwide, rendering it highly possible that PA-containing plants are the most common poisonous plants affecting livestock and humans. PAs require metabolic activation mediated by cytochrome P450 enzymes to generate reactive pyrrolic metabolites that react with cellular proteins and DNA leading to hepatotoxicity and genotoxicity. PAs can also modulate both phase I and phase II metabolizing enzymes, which may alter the metabolic fate of endogenous and exogenous chemicals. Alteration and/or competition of the metabolizing enzymes by PAs upon the co-administered herbal medicines or drugs can potentially result in serious clinical and toxicological consequences through decreased pharmacological activities or increased toxic effects.     

Dose-response relationship in intoxication by the pyrrolizidine alkaloid monocrotaline

Rats develop pulmonary hypertension over a 2-wk period of continuous ingestion of monocrotaline dissolved in drinking water (20 mg/l). The relationship between monocrotaline concentration and duration of exposure was investigated by giving male rats (initial body weight 100 g) monocrotaline in drinking water (5, 10, 20, 40, or 60 mg/l) for 0, 1, 2, 4, 6, 10, or 20 d. Rats were killed 20 d after initiating treatment, and increased lung and right ventricular to body weight ratios were measured as indices of pulmonary hypertension. The accumulative dose of monocrotaline delivered over a 10-d period using a drinking water concentration of 10 mg/l (18 mg/kg) produced the same degree of right ventricular hypertrophy and lung weight increases as the doses ingested over a 4-d period by rats consuming 20 or 40 mg/l monocrotaline water (14 and 29 mg/kg). Phenobarbital pretreatment did not substantially alter the time course of toxicity induced with 20 mg/l monocrotaline water. Ingestion of 60 mg/l monocrotaline water for 1 d (11 mg/kg) resulted in right ventricular hypertrophy at 20 d. Since accumulative doses of less than 11 mg/kg did not produce toxicity and all doses greater than 14 mg/kg did, this range may be considered a threshold for inducing toxicity. However, organ weight increases following threshold exposures reversed over a 4-wk period. Increases in the wall thickness of pulmonary arteries correlated with the development of right ventricular hypertrophy. Pulmonary inflammation was not an early response to monocrotaline administration, since there was no change in the proportion of cell types recovered in lung lavage fluid during the first 6 d of monocrotaline treatment.     

Species differences in the in vitro metabolic reduction of the amphetamine metabolite, 1-phenyl-2-propanone

Marked differences were observed, in the ability of fortified 9000 g liver homogenate supernatants from three species to reduce 1-phenyl-2-propanone to the corresponding alcohol. This metabolic keto-reduction was negligible in homogenates from the rat and extensive in the rabbit; guinea-pig liver homogenates had intermediate ability. Metabolic oxidation of 1-phenyl-2-propanol was negligible in all three species. The amount of deamination of amphetamine and of N-n-propylamphetamine was approximately equal, in vitro, in rats and guinea-pigs but two to three times greater in liver homogenates from rabbits. Approximately three times more deaminated products were formed from the in vitro metabolism of N-n-propylamphetamine than from amphetamine metabolism by all three species.     

Metabolic activation of the tumorigenic pyrrolizidine alkaloid, riddelliine, leading to DNA adduct formation in vivo

Riddelliine is a representative naturally occurring genotoxic pyrrolizidine alkaloid. We have studied the mechanism by which riddelliine induces hepatocellular tumors in vivo. Metabolism of riddelliine by liver microsomes of F344 female rats generated riddelliine N-oxide and dehydroretronecine (DHR) as major metabolites. Metabolism was enhanced when liver microsomes from phenobarbital-treated rats were used. Metabolism in the presence of calf thymus DNA resulted in eight DNA adducts that were identical to those obtained from the reaction of DHR with calf thymus DNA. Two of these adducts were identified as DHR-modified 7-deoxyguanosin-N(2)-yl epimers (DHR-3'-dGMP); the other six were DHR-derived DNA adducts, but their structures were not characterized. A similar DNA adduct profile was detected in the livers of female F344 rats fed riddelliine, and a dose-response relationship was obtained for the level of the total (eight) DHR-derived DNA adducts and the level of the DHR-3'-dGMP adducts. These results suggest that riddelliine induces liver tumors in rats through a genotoxic mechanism and the eight DHR-derived DNA adducts are likely to contribute to liver tumor development.     

Model systems for detecting the hepatic toxicity of pyrrolizidine alkaloids and pyrrolizidine alkaloid N-oxides

Indicine N-oxide (INO) is a pyrrolizidine alkaloid (PA) with antitumor activity in animals and humans. Prior studies showed that despite the known hepatic toxicity of the PAs, INO did not produce hepatic toxicity in animals but caused unpredictable lethal hepatic toxicity in humans. In this study we have attempted to find a model system for predicting the hepatotoxic potential of antitumor PAs. Primary cultures of rat hepatocytes showed toxicity only with the most hepatotoxic PAs such as lasiocarpine, but did not detect toxicity with other PAs. Subchronic intraperitoneal administration of PAs to weanling rats and adult mice produced, in surviving animals, hepatic megalocytosis and centrilobular necrosis with heliotrine (H) and 9-O-(R(-)-2-(4'-chlorophenyl)-2-hydroxybutyryl)retronecine N-oxide (RC1NO) but only megalocytosis with INO. Thus, despite previous reports, weanling rats offered no advantage over adult mice for detecting significant hepatic toxicity with PAs. Phenobarbital pretreatment of the mice did not increase the hepatic toxicity of any of the PAs. Subchronic oral administration of PAs to adult mice produced hepatic megalocytosis and centrilobular necrosis in surviving animals with H and RC1NO and megalocytosis with INO. Animals that died acutely following oral administration of INO showed hepatic centrilobular necrosis. Administration of several courses of INO intravenously to dogs produced histological evidence of centrilobular hemorrhagic necrosis. It is concluded that there is no single animal model that will predict hepatic toxicity of the type seen in humans with the antitumor PAs. A combination of studies using adult mice and dogs and lethal doses of the PAs offers the best way of detecting potential hepatic toxicity.     

Resistance of the guinea pig to pyrrolizidine alkaloid intoxication

Severe hepatotoxicity following intoxication with pyrrolizidine alkaloids has been observed in most domestic and laboratory animals. The guinea pig has been an unexplained exception to this generalization. Administration of monocrotaline, a pyrrolizidine alkaloid contained in the seed and vegetation of Crotalaria spectabilis, produced no clinical or pathologic alterations in guinea pigs. The in vitro microsomal ability to convert monocrotaline to its N-oxide derivative was present to the same degree in rats and guinea pigs, but the level of dehydrogenation activity in the rat was considerably greater than in the guinea pig. The ability to produce monocrotaline pyrroles in vivo was also much greater in rats than guinea pigs. However, when the pyrrolic metabolites were administered by iv injection into the mesenteric blood supply, both species developed marked hepatic necrosis. Therefore, monocrotaline N-oxides are not converted to toxic metabolites and are not toxic per se. Monocrotaline pyrroles appear to be the only metabolites yet determined which are responsible for the lesions produced by monocrotaline intoxication. The fact that N-oxidase activity, but not dehydrogenase activity, is present in guinea pig hepatic microsomes supports the contention that N-oxides and pyrroles are formed by separate enzymatic pathways during pyrrolizidine poisoning.     

Species differences in the in vitro metabolic reduction of the amphetamine metabolite, 1-phenyl-2-propanone.

Marked differences were observed, in the ability of fortified 9000 g liver homogenate supernatants from three species to reduce 1-phenyl-2-propanone to the corresponding alcohol. This metabolic keto-reduction was negligible in homogenates from the rat and extensive in the rabbit; guinea-pig liver homogenates had intermediate ability. Metabolic oxidation of 1-phenyl-2-propanol was negligible in all three species. The amount of deamination of amphetamine and of N-n-propylamphetamine was approximately equal, in vitro, in rats and guinea-pigs but two to three times greater in liver homogenates from rabbits. Approximately three times more deaminated products were formed from the in vitro metabolism of N-n-propylamphetamine than from amphetamine metabolism by all three species.     

Species differences in the in vitro metabolism of methapyrilene

1. The metabolism of N,N-dimethyl-N'-(2-pyridyl)-N'-(2-thienylmethyl)-1,2- ethanediamine(methapyrilene, I) by liver microsomes from rat, guinea pig, and rabbit has been examined. 2. Methapyrilene-N-oxide, (III), normethapyrilene, (II), 2-thiophene methanol, (VI), 2-thiophene carboxylic acid, (VII), N-(2-pyridyl)-N',N'-dimethylethylenediamine, (IX), and methapyrilene amide, (XIV) were found in all species. 3. N-(2-Thienylmethyl)-2-amino pyridine, (VIII), 2-aminopyridine, (X), and (5-hydroxypridyl)-methapyrilene, (XII), were detected in rat and rabbit only. 4. N-Hydroxynormethapyrilene, (XXI), was tentatively identified by mass spectral fragmentation patterns only in rabbit liver microsomes incubations; however, it was found in 9000 g supernatant fraction incubations of rabbit, rat and guinea pig. 5. The formation of IX and XII was quantitatively more important in the rat than in either rabbit or guinea pig.     

Species differences in the in vitro metabolism of methapyrilene

1. The metabolism of N, N-dimethyl-N'-(2-pyridyl)-N'-(2-thienylmethyl)-1,2- ethanediamine(methapyrilene, I) by liver microsomes from rat, guinea pig, and rabbit has been examined.

2. Methapyrilene-N-oxide, (III), normethapyrilene, (II), 2-thiophene methanol, (VI), 2-thiophene carboxylic acid, (VII), N-(2-pyridyl)-N',N'-dimethylethylenediamine, (IX), and methapyrilene amide, (XIV) were found in all species.

3. N-(2-Thienylmethyl)-2-amino pyridine, (VIII), 2-aminopyridine, (X), and (5-hydroxypridyl)-methapyrilene, (XII), were detected in rat and rabbit only.

4. N-Hydroxynormethapyrilene, (XXI), was tentatively identified by mass spectral fragmentation patterns only in rabbit liver microsomes incubations; however, it was found in 9000?g supernatant fraction incubations of rabbit, rat and guinea pig.

5. The formation of IX and XII was quantitatively more important in the rat than in either rabbit or guinea pig.     

Species differences in the in vitro metabolism of phencyclidine

1. The metabolism of 1-(1-phenylcyclohexyl)-piperidine (phencyclidine or PCP) by liver preparations from cat, monkey, rabbit and rat has been studied.

2. 4-Phenyl-4-piperidinocyclohexanol (I), 1-1-phenylcyclohexyl-4-hydroxy-piperidine (II), N-(5-hydroxypentyl)-1-phenylcyclohexylamine (IX) and 5-(l-phenylcyclohexylamino)-valeric acid (X) were found in all species, but liver preparations of rat and rabbit were much more active than those of cat or monkey in metabolizing PCP.

3. Only rabbit produced 4-(4′-hydroxypiperidino)-4-phenylcyclohexanol (III) in amounts detectable by g.l.c.

4. Mass balance calculations of PCP, I, II, III, IX and X in the cat, monkey and rat indicate that other metabolic pathways not measured in this study are operative.