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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Mutations induced by the carcinogenic pyrrolizidine alkaloid riddelliine in the liver cII gene of transgenic big blue rats

Riddelliine is a naturally occurring pyrrolizidine alkaloid that forms a number of different mononucleotide and dinucleotide adducts in DNA. It is a rodent carcinogen and a potential human hazard via food contamination. To examine the mutagenicity of riddelliine, groups of six female transgenic Big Blue rats were gavaged with 0.1, 0.3, and 1.0 mg riddelliine per kg body weight. The middle and high doses resulted in liver tumors in a previous carcinogenesis bioassay. The animals were treated 5 days a week for 12 weeks and sacrificed 1 day after the last treatment. The liver DNA was isolated for analysis of the mutant frequency (MF) in the transgenic cII gene, and the types of mutations were characterized by sequencing the mutants. A significant dose-dependent increase in MF was found, increasing from 30 x 10(-)(6) in the control animals to 47, 55, and 103 x 10(-)(6) in the low, middle, and high dose groups, respectively. Molecular analysis of the mutants indicated that there was a statistically significant difference between the mutational spectra from the riddelliine-treated and the control rats. A G:C --> T:A transversion (35%) was the major type of mutation in rats treated with riddelliine, whereas a G:C --> A:T transition (55%) was the predominant mutation in the controls. In addition, mutations from the riddelliine-treated rats included an unusually high frequency (8%) of tandem base substitutions of GG --> TT and GG --> AT. These results indicate that riddelliine is a genotoxic carcinogen in rat liver and that the types of mutations induced by riddelliine are consistent with riddelliine adducts involving G:C base pairs.     

Differential metabolism of the pyrrolizidine alkaloid,senecionine, in fischer 344 and sprague-dawley Rats

The pyrrolizidine alkaloids (PAs), contained in a number of traditional remedies in Africa and Asia, show wide variations in metabolism between animal species but little work has been done to investigate differences between animal strains. The metabolism of the PA senecionine (SN) in Fischer 344 (F344) rats has been studied in order to compare to that found in the previously investigated Sprague-Dawley (SD) rats (Drug Metab. Dispos. 17: 387, 1989). There was no difference in the formation of (+/-) 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP, bioactivation) by hepatic microsomes from either sex of SD and F344 rats. However, hepatic microsomes from male and female F344 rats had greater activity in the N-oxidation (detoxication) of SN by 88% and 180%, respectively, when compared to that of male and female SD rats. Experiments conducted at various pH showed an optimum pH of 8.5, the optimal pH for flavin-containing monooxygenase (FMO), for SN N-oxidation by hepatic microsomes from F344 females. In F344 males, however, a bimodal pattern was obtained with activity peaks at pH 7.6 and 8.5 reflecting the possible involvement of both cytochrome P450 (CYP) and FMO. Use of specific inhibitors (SKF525A, 1-benzylimidazole and methimazole) showed that the N-oxide of SN was primarily produced by FMO in both sexes of F344 rats. In contrast, SN N-oxide formation is known to be catalyzed mainly by CYP2C11 rather than FMO in SD rats. This study, therefore, demonstrated that there were substantial differences in the formation of SN N-oxide by hepatic microsomes from F344 and SD rats and that this detoxification is catalyzed primarily by two different enzymes in the two rat strains. These findings suggest that significant variations in PA biotransformation can exist between different animal strains.     

Microsomal formation of a pyrrolic alcohol glutathione conjugate of the pyrrolizidine alkaloid senecionine.

1. Pyrrolizidine alkaloids (PAs) are metabolized primarily to putative dehydroalkaloid (PA pyrrole) metabolites and to PA N-oxide by rat liver microsomal monooxygenases. 2. The dehydroalkaloids are highly reactive and either bind covalentely to tissue nucleophiles or are hydrolysed to the more stable pyrrole, (R,S)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP), and the corresponding necic acid. 3. Addition of glutathione (GSH 1 mM) to incubation mixtures containing rat liver microsomes and the PA senecionine (SN), resulted in the formation of a conjugate of DHP with GSH. 5. The mass spectrum of this DHP-GSH conjugate was identical to that of the chemically-synthesized dehydroretronecine (the R enantiomer of the racemic DHP) and GSH. 6. Only negligible amounts of DHP-GSH conjugate were formed when DHP itself was incubated with GSH at physiological pH. 7. These findings provide strong evidence for the microsomal conversion of SN to a highly reactive metabolite, presumably dehydrosenecionine, which then reacts with GSH to form the DHP-GSH conjugate. 8. It is likely that a similar mechanism is responsible in vivo for the formation of GSH conjugates of DHP from SN and other PAs.     

Toxicology and carcinogenesis studies of riddelliine (CAS No. 23246-96-0) in F344/N rats and B6C3F1 mice (gavage studies)

Riddelliine belongs to a class of toxic pyrrolizidine alkaloids and is isolated from plants of the genera Crotalaria, Amsinckia, and Senecio that grow in the western United States. Cattle, horses, and sheep that ingest these plants succumb to their toxic effects. Riddelliine residues have been found in meat, milk, and honey, and the plants may contaminate human food sources. Riddelliine was nominated for study by the Food and Drug Administration because of its potential for human exposure and its economic impact on the livestock industry and because the toxicity of other pyrrolizidine alkaloids suggests riddelliine may be carcinogenic. Male and female F344/N rats and B6C3F1 mice received riddelliine (approximately 92% pure) by gavage. Female rats and male and female mice were dosed for 2 years; due to high mortality, the study in male rats was terminated at week 72. In vitro genetic toxicology studies were conducted in Salmonella typhimurium and in cultured Chinese hamster ovary (CHO) cells. In addition, riddelliine was evaluated in vivo for induction of micronuclei in mouse bone marrow and peripheral blood erythrocytes and for induction of S-phase DNA synthesis and unscheduled DNA synthesis in the liver of rats and mice. Riddelliine-induced DNA adduct levels were determined in liver tissue obtained from female rats admininstered riddelliine for 3 or 6 months. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female rats were administered 0 or 1 mg riddelliine/kg body weight in sodium phosphate buffer by gavage 5 days per week; additional groups of 50 female rats received 0.01, 0.033, 0.1, or 0.33 mg/kg. A wide dose range was used in female rats to better characterize the dose-response curve. Females were dosed for 105 weeks; due to high mortality, male rats were terminated at week 72. All but three 1 mg/kg males died before week 70, and all 1 mg/kg females died before week 97. Mean body weights of 1 mg/kg males and females were less than those of the vehicle controls throughout most of the study. The only clinical finding related to riddelliine administration was a general debilitation of the animals prior to death. Hemangiosarcomas were present in the liver of 86% of males and 76% of females in the 1 mg/kg groups, and this neoplasm was considered the cause of the large number of early deaths in these groups. The incidences of hepatocellular adenoma and mononuclear cell leukemia in 1 mg/kg males and females were significantly increased. Nonneoplastic lesions related to riddelliine treatment occurred in the liver and kidney of males and females. Analyses of liver tissue from female rats treated with riddelliine for 3 or 6 months yielded eight DNA adducts; these were the same as DNA adducts formed in vitro by the metabolism of riddelliine by human liver microsomes in the presence of calf thymus DNA. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female mice were administered riddelliine in sodium phosphate buffer by gavage at doses of 0 or 3 mg/kg, 5 days per week, for 105 weeks; additional groups of 50 male mice received 0.1, 0.3, or 1 mg/kg for 105 weeks. A wide dose range was used in male mice to better characterize the dose-response curve. Survival of males and females administered 3 mg/kg was significantly less than that of the vehicle controls. Mean body weights of 3 mg/kg mice were less than those of the vehicle controls throughout most of the study. Hemangiosarcomas of the liver were present in 62% of males in the 3 mg/kg group. The incidences of hepatocellular neoplasms occurred with negative trends in male mice and were significantly decreased in 3 mg/kg females. The incidences of alveolar/bronchiolar neoplasms in 3 mg/kg females were significantly increased. Nonneoplastic lesions related to riddelliine administration occurred in the liver and kidney of males and females and in the lung and arteries (multiple tissues) of females. GENETIC TOXICOLOGY: Riddelliine was mutagenic in S. typhimurium strain TA100 with, but not without, S9 activation; no significant mutagenic activity was detected in strain TA98 or TA1535,ed in strain TA98 or TA1535, with or without S9. A small, dose-related increase in mutant colonies seen in strain TA97 with S9 was judged to be equivocal. Riddelliine induced sister chromatid exchanges in cultured CHO cells with and without S9. Chromosomal aberrations were induced in CHO cells only in the presence of S9. Following 4 or 13 weeks of daily gavage treatment with riddelliine, no increases in the frequency of micronucleated erythrocytes were noted in the peripheral blood of male or female B6C3F1 mice. Use of a single intraperitoneal injection protocol, however, produced a small but significant increase in the frequency of micronucleated eryth-rocytes in peripheral blood of male Swiss mice 48 hours after injection; bone marrow analysis 24 hours after injection demonstrated a small but insignificant increase in the frequency of micronuclei. Unscheduled DNA synthesis was detected in cultured hepatocytes from male and female rats and mice following 5 or 30 days of riddelliine treatment by gavage. In addition, an S-phase DNA synthesis was observed in cultured hepatocytes of male and female rats treated for either time period. CONCLUSIONS: Under the conditions of these studies, there was clear evidence of carcinogenic activity of riddelliine in male and female F344/N rats based primarily on increased incidences of hemangiosarcoma in the liver. The increased incidences of hepatocellular adenoma and mononuclear cell leukemia in male and female rats were also considered to be treatment related. There was clear evidence of carcinogenic activity of riddelliine in male B6C3F1 mice based on increased incidences of hemangiosarcoma in the liver. There was clear evidence of carcinogenic activity in female B6C3F1 mice based on increased incidences of alveolar/bronchiolar neoplasms. Administration of riddelliine by gavage resulted in nonneoplastic lesions in the liver and kidney of male and female rats; the liver and kidney of male and female mice; and the lung and arteries (multiple tissues) of female mice. Decreased incidences of hepatocellular neoplasms in male and female mice were related to riddelliine administration.     

Metabolism, genotoxicity, and carcinogenicity of comfrey

Comfrey has been consumed by humans as a vegetable and a tea and used as an herbal medicine for more than 2000 years. Comfrey, however, produces hepatotoxicity in livestock and humans and carcinogenicity in experimental animals. Comfrey contains as many as 14 pyrrolizidine alkaloids (PA), including 7-acetylintermedine, 7-acetyllycopsamine, echimidine, intermedine, lasiocarpine, lycopsamine, myoscorpine, symlandine, symphytine, and symviridine. The mechanisms underlying comfrey-induced genotoxicity and carcinogenicity are still not fully understood. The available evidence suggests that the active metabolites of PA in comfrey interact with DNA in liver endothelial cells and hepatocytes, resulting in DNA damage, mutation induction, and cancer development. Genotoxicities attributed to comfrey and riddelliine (a representative genotoxic PA and a proven rodent mutagen and carcinogen) are discussed in this review. Both of these compounds induced similar profiles of 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-derived DNA adducts and similar mutation spectra. Further, the two agents share common mechanisms of drug metabolism and carcinogenesis. Overall, comfrey is mutagenic in liver, and PA contained in comfrey appear to be responsible for comfrey-induced toxicity and tumor induction.     

Microsomal formation of a pyrrolic alcohol glutathione conjugate of the pyrrolizidine alkaloid senecionine

1. Pyrrolizidine alkaloids (PAs) are metabolized primarily to putative dehydroalkaloid (PA pyrrole) metabolites and to PA N-oxide by rat liver microsomal monooxygenases.

2. The dehydroalkaloids are highly reactive and either bind covalentely to tissue nucleophiles or are hydrolysed to the more stable pyrrole, (R, S)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP), and the corresponding necic acid.

3. Addition of glutathione (GSH 1 mM) to incubation mixtures containing rat liver microsomes and the PA senecionine (SN), resulted in the formation of a conjugate of DHP with GSH.

5. The mass spectrum of this DHP-GSH conjugate was identical to that of the chemically-synthesized dehydroretronecine (the R enantiomer of the racemic DHP) and GSH.

6. Only negligible amounts of DHP-GSH conjugate were formed when DHP itself was incubated with GSH at physiological pH.

7. These findings provide strong evidence for the microsomal conversion of SN to a highly reactive metabolite, presumably dehydrosenecionine, which then reacts with GSH to form the DHP-GSH conjugate.

8. It is likely that a similar mechanism is responsible in vivo for the formation of GSH conjugates of DHP from SN and other PAs.     

3-aminobenzanthrone, a human metabolite of the environmental pollutant 3-nitrobenzanthrone, forms DNA adducts after metabolic activation by human and rat liver microsomes: evidence for activation by cytochrome P450 1A1 and P450 1A2

3-Nitrobenzanthrone (3-NBA) is a suspected human carcinogen found in diesel exhaust and ambient air pollution. The main metabolite of 3-NBA, 3-aminobenzanthrone (3-ABA), was recently detected in the urine of salt mining workers occupationally exposed to diesel emissions. Determining the capability of humans to metabolize 3-ABA and understanding which human enzymes are involved in its activation are important in the assessment of individual susceptibility. We compared the ability of eight human hepatic microsomal samples to catalyze DNA adduct formation by 3-ABA. Using the (32)P-postlabeling method, we found that all hepatic microsomes were competent to activate 3-ABA. DNA adduct patterns with multiple adducts, qualitatively similar to those formed in vivo in rats treated with 3-ABA, were observed. These patterns were also similar to those formed by the nitroaromatic counterpart 3-NBA and which derive from reductive metabolites of 3-NBA bound to purine bases in DNA. The role of specific cytochrome P450s (P450s) in the human hepatic microsomal samples in 3-ABA activation was investigated by correlating the P450-linked catalytic activities in each microsomal sample with the level of DNA adducts formed by the same microsomes. On the basis of this analysis, most of the hepatic microsomal activation of 3-ABA was attributable to P450 1A1 and 1A2 enzyme activity. Inhibition of DNA adduct formation in human liver microsomes by alpha-naphthoflavone and furafylline, inhibitors of P450 1A1 and 1A2, and P450 1A2 alone, respectively, supported this finding. Using recombinant human P450 1A1 and 1A2 expressed in Chinese hamster V79 cells and microsomes of baculovirus-transfected insect cells (Supersomes), we confirmed the participation of these enzymes in the formation of 3-ABA-derived DNA adducts. Moreover, essentially the same DNA adduct pattern found in microsomes was detected in metabolically competent human lymphoblastoid MCL-5 cells expressing P450 1A1 and 1A2. Using rat hepatic microsomes, we showed that both human and rat microsomes lead to the same 3-ABA-derived DNA adducts. Pretreatment of rats with beta-naphthoflavone or Sudan I, inducers of P450 1A1 and 1A2, and P450 1A1 alone, respectively, significantly stimulated the levels of 3-ABA-derived DNA adducts formed by rat liver microsomes. Utilizing purified rat recombinant P450 1A1, the participation of this enzyme in DNA adduct formation by 3-ABA was corroborated. In summary, our results strongly suggest a genotoxic potential of 3-ABA for humans. Moreover, 3-ABA is not only a suitable biomarker of exposure to 3-NBA but may also directly contribute to the high genotoxic potential of 3-NBA.     

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.     

Metabolism,Genotoxicity, annd Carcinogenicity of Comfrey

Comfrey has been consumed by humans as a vegetable and a tea and used as an herbal medicine for more than 2000 years. Comfrey, however, produces hepatotoxicity in livestock and humans and carcinogenicity in experimental animals. Comfrey contains as many as 14 pyrrolizidine alkaloids (PA), including 7-acetylintermedine, 7-acetyllycopsamine, echimidine, intermedine, lasiocarpine, lycopsamine, myoscorpine, symlandine, symphytine, and symviridine. The mechanisms underlying comfrey-induced genotoxicity and carcinogenicity are still not fully understood. The available evidence suggests that the active metabolites of PA in comfrey interact with DNA in liver endothelial cells and hepatocytes, resulting in DNA damage, mutation induction, and cancer development. Genotoxicities attributed to comfrey and riddelliine (a representative genotoxic PA and a proven rodent mutagen and carcinogen) are discussed in this review. Both of these compounds induced similar profiles of 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-derived DNA adducts and similar mutation spectra. Further, the two agents share common mechanisms of drug metabolism and carcinogenesis. Overall, comfrey is mutagenic in liver, and PA contained in comfrey appear to be responsible for comfrey-induced toxicity and tumor induction.     

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.     

Tamoxifen-DNA adducts formed by alpha-acetoxytamoxifen N-oxide

DNA adduct formation is assumed to be a major carcinogenic event, leading to the development of endometrial cancer in breast cancer patients taking tamoxifen and healthy women enrolled in a tamoxifen chemopreventive trial. To determine whether DNA adducts were formed by tamoxifen, trans- and cis-alpha-acetoxytamoxifen N-oxides were synthesized as model-activated forms via major tamoxifen metabolites, tamoxifen N-oxide and alpha-hydroxytamoxifen N-oxide. When alpha-acetoxytamoxifen N-oxide was reacted with human DNA, at least three DNA adducts were detected by (32)P-postlabeling coupled with HPLC. The total amount of DNA adducts formed by trans-alpha-hydroxytamoxifen N-oxide was 1.5-fold higher than that formed by the cis form. Both trans- and cis-alpha-acetoxytamoxifen N-oxide reacted with 2'-deoxyguanosine, resulting in the formation of three adducts (fr-1, fr-2-1, and fr-2-2). These products were studied using mass spectroscopy and proton magnetic resonance spectroscopy. fr-1 was identified as a mixture of the epimers of trans-alpha-(N(2)-deoxyguanosinyl)tamoxifen N-oxide. fr-2-1 and fr-2-2 were determined to be epimers of cis-alpha-(N(2)-deoxyguanosinyl)tamoxifen N-oxide.     

GC/MS/MS detection of pyrrolic metabolites in animals poisoned with the pyrrolizidine alkaloid riddelliine

Pyrrolic metabolites from pyrrolizidine alkaloids (PAs) were detected in liver and dried blood samples using a gas chromatography/tandem mass spectrometry (GC/MS/MS) selected product-ion-monitoring method. A calibration curve was constructed using a protein-metabolite conjugate spiked into dried bovine blood. These spiked samples served as a model for tissues from animals poisoned by the toxic metabolite of PAs. Tissue samples from pigs fed various amounts of the PA alkaloid riddelliine (from Senecio riddellii) were analyzed for pyrrolic metabolites, and the results were applied to the calibration curve to provide a measure of the degree of PA poisoning. Pyrrolic metabolites were detected in liver and blood samples of all poisoned animals at levels between 2 and 64 ppm. Although differences in metabolite levels could be discerned under the reported experimental conditions, the amount detected did not correlate with the dose of riddelliine given; and livers fixed with formalin gave greatly reduced recovery than those same livers either frozen or freeze dried.