Metabolic Formation of DHP-Derived DNA Adducts from a

Apr 29, 2004 - The Ligularia hodgsonnii Hook plant, an antitussive traditional Chinese medicine, was found to contain otonecine type PAs with clivorin...
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Chem. Res. Toxicol. 2004, 17, 702-708

Metabolic Formation of DHP-Derived DNA Adducts from a Representative Otonecine Type Pyrrolizidine Alkaloid Clivorine and the Extract of Ligularia hodgsonnii Hook Qingsu Xia,† Ming W. Chou,† Ge Lin,*,‡ and Peter P. Fu*,† Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, Arkansas 72079, and Department of Pharmacology, The Chinese University of Hong Kong, Shatin, Hong Kong, Special Administrative Region Received June 18, 2003

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-hydroxymethyl5H-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 DHPderived 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.

Introduction Pyrrolizidine alkaloids (PAs) have been found in more than 6000 plant species of different families distributed in many geographical regions in the world (1-4). PAcontaining plants are probably one of the most common poisonous plants affecting livestock, wildlife, and humans (1-12). Because hepatotoxic and tumorigenic PAs have been detected in dietary supplements, herbal tea, and herbal plants, human exposure to PAs has been a public health concern (1-4, 7-9). The International Program on Chemical Safety has determined that PAs are a threat to human health and safety (6, 7). Several western countries have established regulations to limit the use of PA-containing herbal plants and dietary supplements (2, 12). The Federal Health Department of Germany has mandates that the PA-containing herbal plants “may be sold and used only if daily internal exposure to no more than 1 µg per day for no more than six weeks a year” (12). The U.S. Food and Drug Administration has requested the withdrawal of comfrey dietary supplements, which contain tumorigenic PAs, from the market (13). Although PA carcinogenesis has been studied for several decades, the mechanisms by which PAs induce * To whom correspondence should be addressed. (P.P.F.) Tel: 870543-7207. Fax: 870-543-7136. E-mail: [email protected]. (G.L.) Tel: 852-2609-6824. Fax: 852-2603-5139. E-mail: [email protected]. † National Center for Toxicological Research. ‡ The Chinese University of Hong Kong.

tumors are not well-understood. We have recently determined that riddelliine, a representative tumorigenic retronecine type PA, induced liver tumors in rats and mice through a genotoxic mechanism (14). The metabolism of riddelliine yielded DHP,1 which then formed eight DHP-derived DNA adducts in vivo and in vitro (14-16). A strong correlation was found between the levels of DHP-derived DNA adducts in different tissues and cell types and the cancer incidence in these tissues, which indicates that the DHP-derived DNA adducts are responsible for the riddelliine-induced liver tumorigenicity (17). We have also determined that the metabolic pattern and DNA adduct profiles from human liver microsomes are similar to those formed in rat liver in vitro and in vivo (18). The kinetic parameters, such as Vmax and Km, obtained from human liver microsomal metabolism of riddelliine are also comparable to those from rat liver microsomal metabolism. Taken together, these results strongly indicate that mechanistic studies of riddelliine 1 Abbreviations: DHP, (()-6,7-dihydro-7-hydroxy-1-hydroxymethyl5H-pyrrolizine; DHR, dehydroretronecine [(-)-6,7-dihydro-7-hydroxy1-hydroxymethyl-5H-pyrrolizine]; ATP, adenosine 5′-triphosphate; MN, micrococcal nuclease; SPD, spleen phosphodiesterase; 3′-dGMP, 2′deoxyguanosine-3′-monophosphate; DHR-3′-dGMP adducts (I and II), 3′-monophosphate of 7-(deoxyguanosin-N2-yl)dehydrosupinidine; DHR3′,5′-dG-bisphosphate adducts (I and II), 3′,5′-bisphosphate of 7-(deoxyguanosin-N2-yl)dehydrosupinidine; 3′-dAMP, 2′-deoxyadenosine-3′-monophosphate; PNK, cloned T4 polynucleotide kinase; TAO, triacetyloleandomycin; NTP, National Toxicology Program; NCTR, National Center for Toxicological Research.

10.1021/tx030030q CCC: $27.50 © 2004 American Chemical Society Published on Web 04/29/2004

Clivorine and the Extract of Ligularia hodgsonnii Hook

with experimental rodents (15) are highly relevant to humans and suggest that riddelliine can be genotoxic to humans and that the genotoxic mechanism is mediated by DHP-derived DNA adduct formation (18). Currently, it is not known whether this mechanism is general to PAs of other types, such as the otonecine type PAs. Clivorine, which has been shown to induce tumors in rats, is a prototype for studying the metabolism, hepatotoxicity, and tumorigenicity of the otonecine type PAs (19-25). The herbal plant derived from Ligularia hodgsonnii Hook, an antitussive traditional Chinese medicine, has been recently found by Lin and co-workers (2001) to contain at least two otonecine type PAs with clivorine being predominant. In this study, we reported that metabolism of clivorine and the extract of this PAcontaining plant by F344 rat liver microsomes results in the formation of DHP. When incubations were conducted in the presence of calf thymus DNA, the same eight DHP-derived DNA adducts as those formed by riddelliine (14-16) were found. This represents the first case in which the same exogenous DNA adducts are formed from two different types (retronecine type and otonecine type) of the same class of chemical carcinogens.

Materials and Methods Materials. Clivorine, with a purity higher than 99% determined by HPLC analysis, was prepared as previously described (21). Troleandomycin (TAO), ketoconazole, calf thymus DNA (sodium salt, type I), 3′-dGMP (sodium salt), glucose-6-phosphate, glucose-6-phosphate dehydrogenase, nicotinamide adenine dinucleotide phosphate (NADP+), nuclease P1, MN, SPD, bicine, spermidine, and dithiothreitol were purchased from the Sigma Chemical Co. (St. Louis, MO). PNK was obtained from U.S. Biochemical Corp. (Cleveland, OH). [γ-32P]ATP (sp. act. >7000 Ci/mmol) was purchased from ICN Biomedicals, Inc. (Costa Mesa, CA). 4-(Dimethylamino)pyridine and 4-dimethylaminobenzaldehyde, used for the Ehrlich assay, were purchased from Aldrich Chemical Co. (Milwaukee, WI). All solvents used were HPLC grade. Male F344 rats were obtained from the NCTR breeding colony as weanlings. Liver microsomes of male F344 rats were prepared as previously described (26). Protein concentrations were determined using a protein assay based on the Bradford method using a Bio-Rad protein detection kit (Bio-Rad Laboratories, Hercules, CA). Preparation of L. hodgsonnii Hook Extract. L. hodgsonnii Hook used as an antitussive traditional Chinese medicine was bought from a Chinese herbal plant wholesale market in Chengdu, Sichuan, China. One kilogram of air-dried plant was ground into fine powder. Following the published procedure (21, 27) with modifications, the plant powder was extracted with 0.05 M hydrochloric acid at 60 °C for 3 h. The insoluble materials were filtered, and the filtrate was partitioned twice with an equal volume of ethyl acetate. The aqueous phase was collected, made basic (pH ∼ 8) with 6 N ammonium hydroxide, and extracted with chloroform three times. The organic layer was collected, the solvent was removed under reduced pressure, and the resultant residue was analyzed by reverse phase HPLC employing a Prodigy 5 µm ODS column (4.6 mm × 250 mm, Phenomenex, Torrance, CA) eluted isocratically with 20 mM ammonium acetate buffer (pH 7) in methanol (1/1; v/v) at 1 mL/min. Metabolism of Clivorine and Extract of L. hodgsonnii Hook by Rat Liver Microsomes. The metabolism of clivorine by male F344 rat liver microsomes was conducted in a 1.0 mL incubation volume containing 100 mM sodium phosphate buffer (pH 7.6), 5 mM magnesium chloride, 1 mM NADP+, 8 mM glucose 6-phosphate, 2 units of glucose 6-phosphate dehydrogenase, 0.2 mM clivorine, and 2 mg of microsomal protein at

Chem. Res. Toxicol., Vol. 17, No. 5, 2004 703 37 °C for 30 min. After incubation, the mixture was centrifuged at 105 000g for 30 min at 4 °C to remove microsomal proteins. The supernatant was collected, and the metabolites present in the incubation mixture were separated by reverse phase HPLC following the conditions previously described by Cui and Lin with modifications (23). It involved the use of two injectors and two HPLC columns, with a Prodigy 5 µm ODS guard column (4.6 mm × 30 mm, Phenomenex) connected between the two injectors and a Prodigy 5 µm ODS analytical column (4.6 mm × 250 mm) following the second injector. The second injector was first connected to a waste bottle (instead of to the second column). The incubation mixture was eluted through the guard column isocratically with 20 mM ammonium acetate buffer (pH 7) for 5 min at 1 mL/min to wash out the water soluble impurities. The second injector was then switched to the position connecting to the second (analytical) column, and the column was eluted with a 15 min linear gradient from 20 mM ammonium acetate buffer (pH 7.0) to 50% methanol in the buffer followed by another linear gradient to 80% methanol in buffer in a period of 15 min. Metabolism of Clivorine by Rat Liver Microsomes in the Presence of P450 3A Enzyme Inhibitor. The metabolism of clivorine by male F344 rat liver microsomes in the presence of an enzyme inhibitor, TAO or ketoconazole, was similarly conducted as described above without inhibitor concentrations being optimized. One milliliter of incubation mixture containing 100 mM sodium phosphate buffer (pH 7.6), 5 mM magnesium chloride, 1 mM NADP+, 8 mM glucose 6-phosphate, 2 units of glucose 6-phosphate dehydrogenase, and 1 mg of microsomal protein was preincubated with 40 nmol of TAO or 10 nmol of ketoconazole (in DMSO) at 37 °C for 10 min. Then, clivorine was added and the resulting incubation mixture was incubated at 37 °C for 30 min. Control incubations in the presence of DMSO were also conducted in parallel. Metabolism of Clivorine, Riddelliine, and Extract of L. hodgsonnii Hook in the Presence of Calf Thymus DNA. The metabolism of clivorine (0.2 and 0.5 mM, respectively) in the presence of calf thymus DNA (1.0 mg) was conducted in a 1 mL incubation volume with conditions identical to those for metabolism. After incubation, the reaction was cooled with ice water and then sequentially extracted with 1.0 mL of phenol, 1.0 mL of phenol/chloroform/isoamyl alcohol (v/v/v, 25/24/ 1), and 1.0 mL of chloroform/isoamyl alcohol (v/v, 24/1). The DNA in the aqueous phase was precipitated by adding 0.1 mL of 5 M sodium chloride followed by an equal volume of cold ethanol and washed three times with 70% ethanol. After the DNA was redissolved in 300 µL of distilled water, the DNA concentration and purity were analyzed spectrophotometrically, and DNA was stored at -78 °C prior to 32P-postlabeling/HPLC analysis. The metabolism of L. hodgsonnii Hook extract in the presence of calf thymus DNA was similarly conducted. For comparison, identification, and quantification of the DHP-derived DNA adducts, microsomal metabolism of riddelliine at concentration of 0.2 and 0.5 mM, respectively, in the presence of calf thymus DNA was also conducted in parallel. Reaction of Clivorine with Calf Thymus DNA. To serve as a negative control, a DNA sample was prepared by reaction of purified calf thymus DNA (1 mg/mL, dissolved in distilled water) with clivorine (0.2 mM) at 37 °C for 30 min. After incubation, the resulting DNA was similarly isolated and quantified as described above and then analyzed by 32P-postlabeling/ HPLC. 32P-Postlabeling/HPLC Analysis of DHP-Derived DNA Adducts. Following previously established procedures for 32P-postlabeling/HPLC analysis (14, 28), 10 µg of the DNA (in 10 µL of distilled water) from the incubations with riddelliine, clivorine, and L. hodgsonnii Hook extract was enzymatically hydrolyzed at 37 °C for 20 min with 78 milliunits (mU) of MN and 4 mU of SPD in a 20 µL solution of 20 mM sodium succinate and 10 mM calcium chloride (pH 6) followed by nuclease P1 enrichment at 37 °C for 20 min. The resulting incubation

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Figure 1. Reversed phased HPLC chromatogram of PAs present in the extract of L. hodgsonnii Hook. HPLC analysis was conducted on a Prodigy 5 µm ODS column (4.6 mm × 250 mm; Phenomenex) eluted isocratically with 20 mM ammonium acetate buffer (pH 7) in methanol (1/1; v/v) at 1 mL/min. mixture was evaporated under reduced pressure, reconstituted into 10 µL of distilled water, and incubated with 10 µL of PNK mix containing 100 µCi of [γ-32P]ATP (sp. act., 7000 Ci/mmol), 12 units of PNK, and 2 µL of 10 × PNK buffer (200 mM bicineNaOH, pH 9.6, 100 mM dithiothreitol, 100 mM MgCl2, and 10 mM spermidine) at 37 °C for 40 min. The 32P-labeled mixture was injected onto a Prodigy 5 µm ODS column (4.6 mm × 250 mm, Phenomenex) and eluted isocratically with 20 mM NaH2PO4 (pH 4.5) for 10 min, followed by a 60 min linear gradient of 20 mM NaH2PO4 (pH 4.5) to 15% methanol in 20 mM NaH2PO4. The HPLC flow rate was 1.0 mL/min, and the scintillation fluid flow rate was 3.0 mL/min. To avoid interference by the high radioactivity of the free 32P and the unreacted [γ-32P]ATP, the on-line FLO-ONE radioactivity detector (Radiomatic Instruments, Tampa, FL) was equipped with a diverter, and the eluent from the first 40 min was diverted from the radioactivity detector. For the comparison and identification of the DHP-derived DNA adducts in the samples, DNA from the reaction of DHR (an enantiomeric form of DHP) and calf thymus DNA was 32P-postlabeled and analyzed by HPLC in parallel using a previously established procedure (28). For quantification of each sample, the two epimeric DHP-3′-dGMP synthetic standards in amounts that closely matched the range of modification in the liver DNA samples were also analyzed in parallel. To serve as a negative control, the DNA from reaction of clivorine with calf thymus DNA in the absence of microsomes was also subjected to 32P-postlabeling/HPLC analysis. Instrumentation. A Waters HPLC system consisting of a model 600 controller, a model 996 photodiode array detector, and pump was used for the separation and purification of DHPderived DNA adducts. For radiochromatography analysis of the 32P-postlabeled reaction mixtures, an HPLC system with online detection of radioactivity was conducted consisting of a solvent gradient programer (Waters model 680), two HPLC pumps (Waters 510), and a radiochromatography detector (Hewlett-Packard FLO-ONE/Beta A-500, Radiomatic Instruments) equipped with a diverter and an autosampler (Waters 717).

Results Confirmation of the Presence of PAs in the L. hodgsonnii Hook Extract. An extract obtained from PA-containing L. hodgsonnii Hook herbal plant was prepared according to the published procedure (21) with

modifications (see Materials and Methods, 27). The presence of PAs in the extract was initially evidenced by the Ehrlich assay performed with TLC analysis (data not shown). The PAs in the extract were then confirmed by reversed phase HPLC (Figure 1), UV-visible absorption spectral (data not shown), and mass spectral (Figure 2) analyses and by comparison of these data with those of authentic samples (21). The chromatographic peak eluted at 8.9 min had the mass spectrum with mass ions at m/z 406 ([M + H]+), 346 (loss of an acetate and a molecule of water), 168, and 150. The material contained in the chromatographic peak that eluted at 14.6 min had a similar mass spectral pattern, with the mass ions at m/z 466 ([M + H]+), 404 (loss of an acetate and a molecule of water), 364 (loss of two acetate and a molecule of water), 168, and 150. The fragment ions at m/z 150 and 168 are present in both mass spectra and are derived from the otonecine base (Figure 2). Thus, the materials contained in the two chromatographic peaks eluted at 8.9 and 14.6 min were identified as clivorine and ligularine, respectively, and both compounds are otonecine type PAs. On the basis of the chromatographic peak areas, the percentage of clivorine and ligularine present in the extract was determined to be 95.7 and 4.3%, respectively, indicating that clivorine was the predominant PA present in this herbal extract. Metabolism of Clivorine and Extract of L. hodgsonnii Hook. The metabolites of clivorine were analyzed by reversed phase HPLC. A typical HPLC chromatogram is shown in Figure 3A. By comparison of HPLC retention time, UV-visible absorption, and mass spectral data (not shown) with those of the standards, the chromatographic peak eluting at 32.5 min was determined to be the intact clivorine, and the peak eluting at 19.7 min was identified as DHP. Metabolism of L. hodgsonnii Hook extract by rat liver microsomes was conducted and analyzed under similar conditions. The chromatographic peak eluting at 19.7 min was similarly identified as DHP. The unchanged clivorine and ligularine were also determined in the incubated mixture (Figure 3B).

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Figure 3. Reversed phased HPLC analysis of metabolites formed from metabolism of (A) clivorine and (B) extract of L. hodgsonnii Hook. HPLC analysis was conducted on a Prodigy 5 µm ODS column (4.6 mm × 250 mm; Phenomenex). For detailed procedures, see the Materials and Methods. Table 1. Levels of Total DHP-Derived DNA Adduct Formed from Male F344 Rat Liver Microsomal Metabolism of Riddelliine and Clivorine in the Presence of Calf Thymus DNAa

compound riddelliine clivorine extract of L. hodgsonnii Hook herbal plant

incubated concn (mM)

total DHP-derived DNA adducts (adducts/106 nucleotides)b

0.2 0.5 0.2 0.5 0.2c

10.0 ( 1.4 13.6 ( 0.9 5.6 ( 0.7 10.1 ( 1.5 5.4 ( 1.3

a For experimental conditions, see the Materials and Methods. Data expressed as mean ( SD from three independent experiments. c On the basis of HPLC analysis, the plant extract contained 0.2 mM clivorine.

b

Figure 2. Mass spectra of the extract components of L. hodgsonnii Hook identified as (A) clivorine and (B) ligularine.

Metabolism of Clivorine by Male F344 Liver Microsomes in the Presence of P450 3A Inhibitor. To determine whether cytochrome P450 3A isozyme is the principal metabolizing enzyme that catalyzes metabolism of clivorine to generate DHP, metabolism of clivorine by male F344 rat liver microsomes was conducted in the presence of ketoconazole or TAO under similar incubation conditions. As compared with the DHP formed from the metabolism without the addition of the enzyme inhibitor, the formation of DHP was 90% reduced by ketoconazole and 85% reduced by TAO, respectively (data not shown). Metabolism of Clivorine and L. hodgsonnii Hook Extract in the Presence of Calf Thymus DNA. We have previously determined that a set of eight DHPderived DNA adducts was formed in liver of rats fed riddelliine in vivo and from metabolism of riddelliine by female F344 rat liver microsomes in vitro (14, 18). Under similar incubation conditions, metabolism of clivorine and L. hodgsonnii Hook extract by rat liver microsomes in the presence of calf thymus DNA was conducted, respectively. To compare the DHP-derived

DNA adduct profile and the level of production, the metabolism of riddelliine was conducted in parallel. As previously described for quantification of DNA adduct formation, reaction of DHP with calf thymus DNA was conducted in parallel (14). Following the experimental conditions previously employed (14), metabolism of riddelliine (at concentration of 0.2 and 0.5 mM, respectively) by male F344 rat liver microsomes in the presence of calf thymus DNA was conducted. As determined by 32P-postlabeling/HPLC analysis, eight DHP-derived DNA adducts were formed (Figure 4A,B). These eight DHP-derived DNA adducts corresponding to the chromatographic peaks eluted at 45.0, 45.1, 48.3, 52.6, 53.6, 56.8, 58.1, and 59.0 min (designated as P1, P2, P3, P4, P5, P6, P7, and P8, respectively). The DNA adducts designated as P4 and P6 are DHP-3′-dGMP adducts, and the other six DHPderived adducts (P1, P2, P3, P5, P7, and P8) were characterized as DHP-derived dinucleotides (3, 14). The levels of the total DHP-derived DNA adducts formed from 0.2 and 0.5 mM riddelliine metabolism were 10.26 ( 1.36 and 13.6 ( 0.94 adducts/106 nucleotides, respectively (Table 1). These results indicate that the total quantity of DHP-derived DNA adducts formed from 0.5 mM riddelliine is about 33% higher than that from 0.2 mM riddelliine.

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Figure 4. 32P-Postlabeling/HPLC analysis of DHP-derived DNA adducts formed from the metabolism of (A) 0.2 mM riddelliine; (B) 0.5 mM riddelliine; (C) 0.2 mM clivorine; (D) 0.5 mM clivorine; and (E) 0.2 mM clivorine present in the extract of L. hodgsonnii Hook. The eight chromatographic peaks eluted at 45.0, 45.1, 48.3, 52.6, 53.6, 56.8, 58.1, and 59.0 min are the identified DHP-derived DNA adducts designated as P1, P2, P3, P4, P5, P6, P7, and P8, respectively.

A similar DHP-derived DNA adduct profile was obtained from metabolism of clivorine (also at concentrations of 0.2 and 0.5 mM, respectively) by male F344 rat liver microsomes in the presence of calf thymus DNA (Figure 4C,D). The levels of the total DHP-derived DNA adducts formed from 0.2 and 0.5 mM clivorine metabolism are 5.6 ( 0.71 and 10.1 ( 1.48 adducts/106 nucleotides, respectively (Table 1). The total quantity of DHPderived DNA adducts formed from 0.5 mM clivorine is about 80% higher than that from 0.2 mM clivorine. However, when clivorine was incubated with calf thymus DNA in the absence of rat liver microsomes (or in the presence of preboiled rat liver microsomes), no DHPderived DNA adducts were determined (data not shown). Furthermore, as shown in Table 1, both clivorine and riddelliine showed a dose-dependent formation of DHPderived DNA adducts. Metabolism of L. hodgsonnii Hook extract, which contained 0.2 mM clivorine, by male F344 rat liver microsomes in the presence of calf thymus DNA also resulted in the formation of the same set of DHP-derived DNA adducts (Figure 4E). The quantity of the resulting DHP-derived DNA adducts formed (5.4 ( 1.3 adducts/ 106 nucleotides) was comparable to that formed from metabolism of 0.2 mM clivorine (5.6 ( 0.71 adducts/106 nucleotides) (Table 1).

Discussion The PAs that exhibit the most potent genotoxicity and tumorigenicity are the macrocyclic diester PAs, namely, the retronecine type, heliotridine type, and otonecine type PAs. We have previously demonstrated that metabolism of riddelliine, a retronecine type PA, by male and female F344 rat liver microsomes in the presence of calf thymus DNA resulted in the formation of a set of eight DHP-

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derived DNA adducts (18). In the present study, metabolism of clivorine, an otonecine type PA, in the presence of calf thymus DNA under similar conditions also produced these eight DHP-derived DNA adducts, in a quantity comparable to that formed from in vitro metabolism of riddelliine. Because riddelliine and clivorine belong to two different tumorigenic types of PAs, our results represent the first report that identical exogenous DNA adducts are formed from two different types of PAs. We have previously determined that these DHP-derived DNA adducts are responsible for riddelliine-induced liver tumorigenicity, which exhibits a dose-response relationship (14). Clivorine has been found to induce liver tumors in rats (25). Thus, the formation of these DNA adducts from clivorine metabolism suggests that these DHPderived DNA adducts are responsible for in part, if not all, clivorine-induced liver tumorigenicity as well as the other genotoxicities. On the basis of the results reported in this study and published previously (14), we hypothesize that these DHP-derived DNA adducts have a potential to be used as biomarkers of PA exposure and PA-induced tumorigenicity. To date, besides riddelliine and clivorine, there are 14 other PAs and one PA N-oxide that have been found to induce tumors in laboratory rodents (29-35). These include (i) nine retronecine type PAs [retronecine (29), retrorsine (30-32), monocrotaline (33, 34), intermedine (35), jacobine (30, 36), lycopsamine (35), seneciphyline (32, 37), symphytine (38, 39), and senecionine (30, 31, 40)]; (ii) two heliotridine type PAs [heliotrine (41) and lasiocarpine (42-45)]; (iii) three otonecine type PAs [senkirkine (37, 39, 40), hydroxysenkirkine (27, 46), and petasitenine (37, 47, 48)]; and (iv) one PA N-oxide [isatidine (retrorsine N-oxide)] (30, 31). Because it is known that metabolism of many of these PAs generated DHP (or its enantiomeric forms, such as DHR) as a principal metabolite, it is possible that metabolism of these tumorigenic PAs would also produce the same set of DHP-derived DNA adducts and that these adducts are responsible for in part, if not all, the tumorigenicity of these PAs. However, this warrants further investigation. On the basis of the present findings, the metabolic scheme for the activation of clivorine to form DHP-derived DNA adducts leading to liver tumors is proposed in Figure 5. The metabolic detoxification pathways leading to glutathione conjugation and the metabolic activation pathway leading to DNA damage and hepatotoxicity (22-24) are also included in Figure 5. Thus, metabolism of clivorine produces a pyrrolic metabolite via oxidative N-demethylation of the necine base followed by ring closure and dehydration. This pyrrolic metabolite, dehydroclivorine, may (i) be hydrolyzed to form the DHP metabolite, (ii) react with glutathione to form the nontoxic glutathione conjugates leading to detoxification, (iii) bind to protein to form the tissue-bound pyrroles responsible for the induction of hepatotoxicity, and (iv) bind to cellular DNA to form the DHP-derived DNA adducts leading to tumors in the liver (Figure 5). Similar to riddelliine metabolism (14, 17, 18), there are two possible pathways that lead to DHP-derived DNA adduct formation: (i) metabolism of clivorine generates dehydroclivorine that binds to DNA followed by hydrolysis, and (ii) dehydroclivorine hydrolyzes to DHP and then DHP binds to DNA. It is currently not known which pathway is predominant.

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Figure 5. Proposed metabolic activation and detoxification pathways of clivorine.

It was also found that DHP formation was 90% inhibited by the potent P450 3A isozyme inhibitor ketoconazole (49, 50) and 85% reduced by the specific human P450 3A4 isoenzyme inhibitor TAO. These results indicate that the DHP formation from rat liver microsomal metabolism of clivorine is primarily catalyzed by P450 3A hepatic metabolizing enzyme. This finding is consistent with our previous report that metabolism of clivorine to reactive pyrrolic species by liver microsomes of male Sprague-Dawley rats and Dunkin-Hartley guinea pigs of both sexes is mainly catalyzed by cytochrome P-450 3A enzyme (24). Lin et al. (24, 51) have recently demonstrated both species and gender differences in the in vitro metabolic activation of clivorine on the generation of reactive pyrrolic metabolites. These metabolism differences were used to interpret why the male rat is significantly more susceptible to clivorine-induced hepatotoxicity than the female rat (51). Similarly, the findings by Lin et al. (24, 51) also suggest that clivorine-induced liver tumorigenicity is species- and gender-dependent. This warrants further investigation. In the present study, we report, for the first time, that DHP-derived DNA adducts were detected from metabolism of the extract of a PA-containing traditional Chinese medicinal plant, L. hodgsonnii Hook. In this extract, two otonecine type PAs were present with a ratio of 96 (clivorine):4 (ligularine); thus, the formation of the same set of eight DHP-derived DNA adducts might be either contributed by both PAs due to the same activation pathway as predicted in Figure 5 or only resulted from the metabolic activation of the predominant clivorine. Further studies are needed to clarify these. Because currently a large number of PA-containing plants have been utilized as herbal medicines, herbal teas, and dietary supplements worldwide (1-4, 7-9), these PAcontaining natural products may pose human health risks. Our detection of DHP-derived DNA adducts from a PA-containing herbal plant upon hepatic metabolism may provide a convenient approach for detecting genotoxic PAs in commercially available natural products.

Acknowledgment. We thank Ms. Shiyou Zhang for the purchase of the L. hodgsonnii Hook plant from China, Dr. Junshi Chen for processing the plant into powder form and shipping it to the investigators, and Dr. Frederick A. Beland for critical review of this manuscript. This research was supported in part by appointment (Q.X.) to the Postgraduate Research Program at the NCTR administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the FDA.

References (1) Mattocks, A. R. (1986) Chemistry and Toxicology of Pyrrolizidine Alkaloids, Academic Press, London, NY. (2) Roeder, E. (1995) Medicinal plants in Europe containing pyrrolizidine alkaloids. Pharmazie 50, 83-98. (3) National Toxicology Program (2001) TR-508 Toxicology and Carcinogensis Studies of Riddelliine (CAS No. 23246-96-0) in F344/N Rats and B6C3F1 Mice (Gavage Studies), NIH Publication No. 01-4442, U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC. (4) International Agency for Research in Cancer (IARC) (1976) Pyrrolizidine alkaloids. IARC Monograph on the Evaluation of Carcinogenic Risk of Chemicals to MansSome Naturally Occurring Substance, International Agency for Research in Cancer, Lyon, France. (5) International Agency for Research in Cancer (IARC) (2002) Senecio Species and Riddelliine. IARC Monograph on the Evaluation of Carcinogenic Risk to HumanssSome Traditional Herbal Medicines, Some Mycotoxins, Naphthalene and Styrene, Vol. 82, pp 153-168, International Agency for Research in Cancer, Lyon, France. (6) International Programme on Chemical Safety (IPCS) (1988) Pyrrolizidine Alkaloids, Environmental Health Criteria 80, WHO, Geneva. (7) International Programme on Chemical Safety (IPCS) (1989) Pyrrolizidine Alkaloids Health and Safety Guide, Health and Safety Criteria Guide 26, WHO, Geneva. (8) Mattocks, A. R. (1968) Toxicity of pyrrolizidine alkaloids. Nature 217, 723-728. (9) Woo, Y.-T., Lai, D. Y., Arcos, J. C., and Argus, M. F. (1988) Chemical Induction of Cancer, Vol. IIIC, pp 212-266, Academic Press Inc., San Diego. (10) Huxtable, R. J. (1980) Herbal teas and toxins: novel aspects of pyrrolizidine poisoning in the United States. Perspect. Biol. Med. 24, 1-14.

708

Chem. Res. Toxicol., Vol. 17, No. 5, 2004

(11) Smith, L. W., and Culvenor, C. C. (1981) Plant sources of hepatotoxic pyrrolizidine alkaloids. J. Nat. Prod. 44, 129-152. (12) Stegelmeier, B. L., Edgar, J. A., Colegate, S. M., Gardner, D. R., Schoch, T. K., Coulombe, R. A., and Molyneux, R. J. (1999) Pyrrolizidine alkaloid plants, metabolism and toxicity. J. Nat. Toxins 8, 95-116. (13) Reuters Medical News at www.medscape.com. (14) Yang, Y. C., Yan, J., Doerge, D. R., Chan, P. C., and Fu, P. P. (2001) Metabolic activation of the tumorigenic pyrrolizidine alkaloid, riddelliine, leading to DNA adduct formation in vivo. Chem. Res. Toxicol. 14, 101-109. (15) Fu, P. P., Xia, Q., Lin, G., and Chou, M. W. (2002) Genotoxic pyrrolizidine alkaloidssmechanisms leading to DNA adduct formation and tumorigenicity. Int. J. Mol. Sci. 3, 945-961. (16) Chou, M. W., Yan, J., Williams, L., Xia, Q., Churchwell, M., Doerge, D. R., and Fu, P. P. (2003) Identification of DNA adducts derived from riddelliine, a carcinogenic pyrrolizidine alkaloid, in vitro and in vivo. Chem. Res. Toxicol. 16, 1130-1137. (17) Chou, M. W., Yan, J., Nichols, J., Xia, Q., Beland, F. A., Chan, P.-C., and Fu, P. P. (2003) Correlation of DNA adduct formation and riddelliine-induced liver tumorigenesis in F344 rats and B6C3F1 mice. Cancer Lett. 193, 119-125. (18) Xia, Q., Chou, M. W., Doerge, D., Kadlubar, F. F., Chan, P.-C., and Fu, P. P. (2003) Human liver microsomal metabolism and DNA adduct formation from the tumorigenic naturally occurring macrocyclic pyrrolizidine alkaloid, riddelliine. Chem. Res. Toxicol. 16, 66-73. (19) Lin, G., Cui, Y. Y., and Hawes, E. M. (1998) Microsomal formation of a pyrrolic alcohol glutathione conjugate of clivorine: Firm evidence for the formation of a pyrrolic metabolite of an otonecinetype pyrrolizidine alkaloid. Drug Metab. Dispos. 26, 181-184. (20) Lin, G., Zhou, K. Y., Zhao, X. G., Wang, Z. T., and But, P. P. H. (1998) Determination of hepatotoxic pyrrolizidine alkaloids by online high performance liquid chromatography mass spectrometry with an electrospray interface. Rapid Commun. Mass Spectrom. 12, 1445-1456. (21) Lin, G., Rose, P., Chatson, K. B., Hawes, E. M., Zhao, X. G., and Wang, Z. T. (2000) Characterization of two structural forms of otonecine-type pyrrolizidine alkaloids from Ligularia hodgsonii by NMR spectroscopy. J. Nat. Prod. 63, 857-860. (22) Lin, G., Cui, Y. Y., and Hawes, E. M. (2000) Characterization of rat liver microsomal metabolites of clivorine, an hepatotoxic otonecine-type pyrrolizidine alkaloid. Drug Metab. Dispos. 28, 1475-1483. (23) Cui, Y. Y., and Lin, G. (2000) Simultaneous determination of clivorine and its four microsomal metabolites by a coupled-column high performance liquid chromatography. J. Chromatogr. A903, 85-92. (24) Lin, G., Cui, Y. Y., and Liu, X. Q. (2002) Species differences in the in vitro metabolic activation of hepatotoxic pyrrolizidine alkaloid, clivorine. Chem. Res. Toxicol. 15, 1421-1428. (25) Kuhara, K., Takanashi, H., Hirono, I., Furuya, T., and Asada, Y. (1980) Carcinogenic activity of clivorine, a pyrrolizidine alkaloid isolated from Ligularia dentata. Cancer Lett. 10, 117-122. (26) Chou, M. W., Wang, B., Von Tungeln, L. S., Beland, F. A., and Fu, P. P. (1987) Induction of rat hepatic cytochromes P-450 by environmental nitropolycyclic aromatic hydrocarbons. Biochem. Pharmacol. 36, 2449-2454. (27) Bartkowski, J. B., Wiedenfeld, H., and Roeder, E. (1997) Quantitative photometric determination of senkirkine in Farfarae folium. Phytochem. Anal. 8, 1-4. (28) Yang, Y.-C., Yan, J., Churchwell, M., Beger, R., Chan, P.-C., Doerge, D. R., Fu, P. P., and Chou, M. W. (2001) Development of a 32P-postlabeling/HPLC method for detection of dehydroretronecine-derived DNA adducts in vitro and in vivo. Chem. Res. Toxicol. 14, 91-100. (29) Schoental, R., and Cavanagh, J. B. (1972) Brain and spinal cord tumors in rats treated with pyrrolizidine alkaloids. J. Natl. Cancer Inst. 49, 665-671. (30) Schoental, R., Head, M. A., and Peacock, P. R. (1954) Senecio alkaloids: Primary liver tumours in rats as a result of treatment

Xia et al.

(31)

(32) (33) (34)

(35)

(36) (37) (38) (39)

(40) (41) (42) (43) (44)

(45) (46) (47)

(48) (49)

(50) (51)

with (1) a mixture of alkaloids from S. jacobaea lin.; (2) retrorsine; (3) isatidine. Br. J. Cancer 8, 458-465. Schoental, R., and Head, M. A. (1957) Progression of liver lesions produced in rats by temporary treatment with pyrrolizidine (senecio) alkaloids, and the effecs of betaine and high casein diet. Br. J. Cancer 11, 535-544. Harris, P. N., and Chen, K. K. (1970) Development of hepatic tumors in rats following ingestion of senecio longilobus. Cancer Res. 30, 2881-2886. Allen, J. R., Hsu, I. C., and Carstens, L. A. (1975) Dehydroretronecine-induced rhabdomyosarcomas in rats. Cancer Res. 35, 997-1002. Shumaker, R. C., Robertson, K. A., Hsu, I. C., and Allen, J. R. (1976) Neoplastic transformation in tissues of rats exposed to monocrotaline or dehydroretronecine. J. Natl. Cancer Inst. 56, 787-790. Schoental, R., Fowler, M. E., and Coady, A. (1970) Islet cell tumors of the pancreas found in rats given pyrrolizidine alkaloids from Amsinckia intermedia Fisch and Mey and from Heliotropium supinum L. Cancer Res. 30, 2127-2131. Cook, J. W., Duffy, E., and Schoental, R. (1950) Primary liver tumours in rats following feeding with alkaloids of senecio jacobaea. Br. J. Cancer 4, 405-410. Hirono, I., Ueno, I., Aiso, S., Yamaji, T., and Haga, M. (1983) Carcinogenic activity of Farfugium japonicum and Senecio cannabifolius. Cancer Lett. 20, 191-198. Hirono, I., Mori, H., and Haga, M. (1978) Carcinogenic activity of symphytum officinale. J. Natl. Cancer Inst. 61, 865-868. Hirono, I., Haga, M., Fujii, M., Matsuura, S., Matsubara, N., Nakayama, M., Furuya, T., Hikichi, M., Takanashi, H., Uchida, E., Hosaka, S., and Ueno, I. (1979) Induction of hepatic tumors in rats by senkirkine and symphytine. J. Natl. Cancer Inst. 63, 469-472. Hirono, I., Mori, H., and Culvenor, C. C. (1976) Carcinogenic activity of coltsfoot, Tussilago farfara L. Gann 67, 125-129. Schoental, R. (1975) Pancreatic islet-cell and other tumors in rats given heliotrine, a monoester pyrrolizidine alkaloid, and nicotinamide. Cancer Res. 35, 2020-2024. Svoboda, D. J., and Reddy, J. K. (1972) Malignant tumors in rats given lasiocarpine. Cancer Res. 32, 908-913. Rao, M. S., and Reddy, J. K. (1978) Malignant neoplasms in rats fed lasiocarpine. Br. J. Cancer 37, 289-293. Rao, M. S., Jago, M. V., and Reddy, J. K. (1983) Effect of calorie restriction on the fate of hyperplastic liver nodules induced by concurrent administration of lasiocarpine and thioacetamide. Hum. Toxicol. 2, 15-26. Svoboda, D. J., and Reddy, J. K. (1974) Laslocarpine-induced, transplantable squamous cell carcinoma of rat skin. J. Natl. Cancer Inst. 53, 1415-1418. Crout, D. H. (1972) Pyrrolizidine and seco-pyrrolizidine alkaloids of Crotalaria laburnifolia L. subspecies eldomae. J. Chem. Soc., Perkin Trans. 1 13, 1602-1607. Hirono, I., Mori, H., Yamada, K., Hirata, Y., and Haga, M. (1977) Carcinogenic activity of petasitenine, a new pyrrolizidine alkaloid isolated from Petasites japonicus Maxim. J. Natl. Cancer Inst. 58, 1155-1157. Furuya, T., Hikichi, M., Iitaka, Y., and Fukinotoxin, A. (1976) A new pyrrolizidine alkaloid from Petasites japonicus. Chem. Pharm. Bull. (Tokyo) 24, 1120-1122. Ghosal, A., Satoh, H., Thomas, P. E., Bush, E., and Moore, D. (1996) Inhibition and kinetics of cytochrome P4503A activity in microsomes from rat, human, and cDNA-expressed human cytochrome P450. Drug Metab. Dispos. 24, 940-947. Ervine, C. M., and Mattew, D. E. (1996) Comparison of ketoconazole and fluconazole an Cytochrome P450 inhibitor. Drug Metab. Dispos. 24, 211-215. Lin, G., Cui, Y. Y., and Liu, X. Q. (2003) Gender differences in mecrosomal metabolic activation of hepatotoxic clivorine in rat. Chem. Res. Toxicol. 16, 768-774.

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