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Chem. Res. Toxicol. 2003, 16, 66-73

Human Liver Microsomal Metabolism and DNA Adduct Formation of the Tumorigenic Pyrrolizidine Alkaloid, Riddelliine Qingsu Xia,† Ming W. Chou,† Fred. F. Kadlubar,† Po-Cheun Chan,‡ and Peter P. Fu*,† National Center for Toxicological Research, Jefferson, Arkansas 72079, and National Institute of environmental Health Sciences, Research Triangle Park, North Carolina 27709 Received August 25, 2002

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.

Introduction Pyrrolizidine alkaloids are naturally occurring heterocyclic compounds that are found worldwide in more than 12 plant families, among which three families, Boraginaceae, Compositae (Asteraceae), and Legumionsae (Fabaceae), contain most toxic pyrrolizidine alkaloids (17). More than 660 pyrrolizidine alkaloids have been identified in over 6000 plants of these three families, and about half of them exhibit toxic activities (7). It has been recognized since the 18th century that many pyrrolizidine alkaloids are highly toxic, causing numerous poisonings in livestocks and humans (1, 2, 4, 5). Pyrrolizidine alkaloids exhibit a variety of genotoxicities, including DNA binding, DNA cross-linking, DNA-protein crosslinking, mutagenicity, and carcinogenicity (1, 2, 6, 7-25). Pyrrolizidine alkaloids require metabolic activation in order to exert their toxicities (2). In general, there are three principal metabolic pathways: (i) hydrolysis of the ester functional group to form the necine bases; (ii) oxidation of the necine bases to the corresponding necine * To whom correspondence should be addressed. National Center for Toxicological Research, Jefferson, AR 72079. Telephone: (870) 5437207. Fax: (870) 543-7136. E-mail: [email protected]. † National Center for Toxicological Research. ‡ National Institute of environmental Health Sciences.

10.1021/tx025605i

N-oxides; and (iii) dehydrogenation of the necine base, through initial hydroxylation at the C-3 or C-8 position to form 3- or 8-hydroxynecine derivatives followed by dehydration, to form the corresponding dehydropyrrolizidine (pyrrolic) derivatives. Metabolism of pyrrolizidine alkaloids to dehydropyrrolizidines is mainly catalyzed by cytochromes P450, specifically both the P450 3A and P450 2B6 enzymes (26-36). Metabolism of pyrrolizidine alkaloids to the corresponding N-oxides is catalyzed by both cytochrome P-450 and flavin-containing monooxygenase (28, 33, 37, 38). Dehydropyrrolizidine metabolites have been shown to be the principle metabolites responsible for genotoxicities of the parent pyrrolizidine alkaloids (2). Because of high instability and easy hydrolysis into (()-6,7-dihydro-7hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP),1 dehydropyrrolizidines cannot be detected in vivo or in vitro. Thus, it is probable that the high genotoxicities of 1 TAO, triacetyloleandomycin (troleandomycin, oleandomycin triacetate); ATP, adenosine 5′-triphosphate; MN, micrococcal nuclease; SPD, spleen phosphodiesterase; DHP, (R/S)6,7-dihydro-7-hydroxy-1hydroxymethyl-5H-pyrrolizine; DHR, dehydroretronecine (or R-6,7dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine; or R-DHP); DHR3′-dGMP adducts (I and II), 3′-monophosphate of 7-(deoxyguanosinN2-yl)dehydrosupinidine; DHR-3′,5′-dG-bisphosphate adducts (I and II), 3′,5′-bisphosphate of 7-(deoxyguanosin-N2-yl)dehydrosupinidine; PNK, cloned T4 polynucleotide kinase; NTP, National Toxicology Program; NCTR, National Center for Toxicological Research.

This article not subject to U.S. Copyright. Published 2003 by the American Chemical Society Published on Web 12/10/2002

Metabolism and DNA Adduct Formation of Riddelliine

dehydropyrrolizidines in vivo are exerted through a combination of dehydropyrrolizidines and DHP (10). Riddelliine, a representative genotoxic pyrrolizidine alkaloid, produced in plants growing in range lands of the western United States (2, 23, 24). Riddelliine exhibits both acute toxicity and a large spectrum of genotoxic activities, including tumorigenicity. Human foodstuffs, such as grains, herbs, milk, honey, herbal teas, and herbal medicine, may be contaminated by pyrrolizidine alkaloids including riddelliine (2, 6). Due to its genotoxicity and potential for human exposure, riddelliine was nominated by the U.S. Food and Drug Administration to the National Toxicology Program (NTP) for genotoxicity and carcinogenicity testing (23, 24). In the NTP 2-year carcinogenicity bioassay, riddelliine induced liver tumors in laboratory male and female rats and male mice (24). As part of the NTP study, we investigated the mechanism by which riddelliine induces liver tumors in laboratory rats and mice (39) and found that metabolism of riddelliine by liver microsomes of F344 female rats generated DHP as a reactive metabolite (6, 39). A set of eight DHP-derived DNA adducts was detected and quantified in liver of rats fed riddelliine (39). Two of these adducts were identified as DHP-derived 7-deoxyguanosin-N2-yl epimers (DHP-3′-dGMP), and the other six were DHP-derived dinucleotide adducts (39, 40). It was also shown that the levels of DNA adduct formation correlated with liver tumor potency (24, 39). Our results suggest that riddelliine induces liver tumors in laboratory male and female rats and male mice through a genotoxic mechanism and the eight DHP-derived DNA adducts are likely to contribute to the liver tumor development (24). To determine whether the mechanistic studies in experimental animals are relevant to humans, in this paper we report the results of metabolism and DNA adduct formation of riddelliine by male and female human liver microsomes and compare the results with those from rat liver microsomal metabolism.

Materials and Methods Materials. Riddelliine was obtained from the NTP (23, 24). DHR and riddelliine N-oxide were prepared following published procedures (39). Troleandomycin (triacetyloleandomycin, TAO), phenobarbital (sodium salt), calf thymus DNA (sodium salt, type I), 2′-deoxyguanosine-3′-monophosphate (sodium salt) (3′dGMP), glucose-6-phosphate, glucose-6-phosphate dehydrogenase, nicotinamide adenine dinucleotide phosphate (NADP+), nuclease P1, micrococcal nuclease (MN), spleen phosphodiesterase (SPD), bicine, spermidine, and dithiothreitol were purchased from the Sigma Chemical Co. (St. Louis, MO). Cloned T4 polynucleotide kinase (PNK) was obtained from U.S. Biochemical Corp. (Cleveland, OH). [γ32P]Adenosine 5′-triphosphate ([γ-32P]ATP) (sp. act. >7000 Ci/mmol) was purchased from ICN Biomedicals, Inc. (Costa Mesa, CA). All other reagents were obtained through commercial sources and were the highest quality available. All solvents used were of HPLC grade. 3′-Monophosphate of 7-(deoxyguanosin-N2-yl)dehydrosupinidine adducts (DHR-3′-dGMP) were prepared in our laboratory (39). DNA from reaction of DHR with calf thymus DNA was prepared as previously described (39). Human livers were obtained from organ donor samples. Four male (no. 4886, 80 years old Caucasian; no. 9504, 62 years old Caucasian; no. 9603, 57 yeas old Caucasian; and no. 9310, 50 years old white) and four female (no. 5807, 69 years old white; no. C055G, 52 years old Caucasian; no. 9603, 50 years old white, and no. 9502, 30 years old African American) liver samples were used for study. All these human liver samples were obtained

Chem. Res. Toxicol., Vol. 16, No. 1, 2003 67 from trauma victims or patients with metastatic disease. Female and male F344 rats were obtained from the National Center for Toxicological Center (NCTR) breeding colony as weanlings. Female and male rat liver microsomes were prepared by combining liver tissues of five female rats and six male F344 rats, respectively. Preparation of Human and Rat Liver Microsomes. Human liver microsomes were prepared and stored as previously described (41). Protein concentrations were determined using a Bio-Rad protein detection kit (Bio-Rad Laboratories, Hercules, CA) based on the Bradford method (42). For comparison, liver microsomes of female and male F344 rats were similarly prepared. Development of Optimal Conditions for Metabolism of Riddelliine by Rat and Human Liver Microsomes. Incubation conditions for rat and human liver microsomal metabolism were optimized to ensure that product formation (DHP or riddelliine N-oxide) was linear with respect to time of incubation and microsomal protein concentration. Microsomal protein concentrations (0.1-2.0 mg/mL of incubation volume), substrate (riddelliine) concentrations (0.05-2.0 mM), and incubation times (5-60 min) were employed for determining optimal incubation conditions. Metabolism of Riddelliine by Rat and Human Liver Microsomes. The metabolism of riddelliine by rat and human liver microsomes of both sexes 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 glucose 6-phosphate dehydrogenase, 0.1 mM riddelliine, and 0.5 mg of microsomal protein at 37 °C for 30 min. After incubation, the incubation mixture was centrifuged at 105000g for 30 min at 4 °C to remove microsomal protein. The supernatant was collected and the metabolite mixture was separated by reversed-phase HPLC employing a Prodigy 5 µm ODS column (10 × 250 mm, Phenomenex, Torrance, CA) eluted isocratically with 20 mM ammonium acetate buffer (pH 7) at 5 mL/min for 10 min followed with a linear gradient from 20 mM ammonium acetate buffer (pH 7.0) to 50% methanol in the buffer for 30 min. For obtaining kinetic parameters of riddelliine metabolism by rat and human liver microsomes, incubations were similarly conducted as described above, with the exception that 1.0 mg of microsomal protein/mL and different substrate concentrations (from 0.1 to 1.6 mM) were used. After incubations, each of the incubation mixtures was centrifuged and the resulting metabolite mixture was separated by reversed-phase HPLC as described above. Values of Vmax and Km of each microsomal incubation results were determined by GraphPad Prism, GraphPad Software, Inc. (San Diego, CA). Metabolism of Riddelliine by Human Liver Microsomes in the Presence of P450 3A4 Enzyme Inhibitor. The metabolism of riddelliine by human liver microsomes of both sexes in the presence of the enzyme inhibitor, TAO, was similarly conducted as described above. First, 0.5 mL of 100 mM sodium phosphate buffer (pH 7.6), containing 5 mM magnesium chloride, 1 mM NADP+, 8 mM glucose 6-phosphate, 1 unit glucose 6-phosphate dehydrogenase, and 0.5 mg microsomal protein was incubated with 20 nmol of TAO (in DMSO) at 37 °C for 30 min, control incubations contained DMSO. After incubation, 0.5 mL of 100 mM sodium phosphate buffer (pH 7.6), containing 5 mM magnesium chloride, 1 mM NADP+, 8 mM glucose 6-phosphate, 1 units glucose 6-phosphate dehydrogenase, and 0.5 µmol of riddelliine was added into above inhibition reaction mixture, and incubated at 37 °C for another 30 min. Metabolism of Riddelliine by Human Liver Microsomes in the Presence of Calf Thymus DNA. The metabolism of riddelliine by human liver microsomes of both sexes 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, 0.1 mM riddelliine, 1.0 mg of purified calf thymus DNA, and 0.5 mg of microsomal protein at

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37 °C for 30 min. After incubation, the reaction was terminated by cooling with ice-water, 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 equal volume of cold ethanol and washed three times with 70% ethanol. After redissolving 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. 32P-Postlabeling/HPLC Analysis of DHP-Derived DNA Adducts. The DHP-derived DNA adducts formed from metabolism of riddelliine by human or rat liver microsomes in the presence of calf thymus DNA were analyzed employing the 32Ppostlabeling/HPLC methodology previously developed in our laboratories (29, 43). Ten micrograms of the DNA dissolved in 10 µL of distilled water was enzymatically hydrolyzed at 37 °C for 20 min with 78 milliunits (mU) of MN and 4 mU of SPD contained in a 20 µL solution of 20 mM sodium succinate and 10 mM of calcium chloride (pH 6). The MN/SPD digested DNA solutions were incubated with nuclease P1 (8 µg, in 4 µL of buffer containing 0.24 M sodium acetate and 2 mM ZnCl2 at pH 5) at 37 °C for 20 min to remove normal 3′-monophosphate of 2′-deoxyribonucleosides. The resulting incubation mixture was then evaporated under reduced pressure, redissolved in 10 µL of distilled water and incubated with 10 µL of PNK mixture 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 µ ODS column (Phenomenex, 4.6 × 250 mm) and eluted isocratically with 20 mM sodium phosphate buffer (pH 4.5) for 10 min, followed by a 60-min linear gradient of 20 mM sodium phosphate buffer (pH 4.5) to 15% methanol in 20 mM sodium phosphate buffer. 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. 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 both radioactivity and UV absorbance was conducted consisting of a solvent gradient programer (Waters Model 680), two HPLC pumps (Waters 510), a UV detector (Waters Model 440), a radio-chromatography detector (HewlettPackard FLO-ONE /Beta A-500) equipped with a diverter and an autosampler (Waters 717).

Results Rat and Human Liver Microsomal Metabolism of Riddelliine. To compare riddelliine metabolism profiles between human and rat liver microsomal metabolism, optical incubation conditions were determined by incubations with different concentrations of microsomal protein (0.1-2.0 mg/mL of incubation volume) and substrate (riddelliine) (0.05-2.0 mM), and with different incubation times (5-60 min). Under the established conditions, product formation (DHP and riddelliine N-oxide) was linear with respect to time of incubation and microsomal protein concentration. After incubation, the metabolism products were separated by reversed-phase HPLC. Figure 1A shows the reversed-phase HPLC profile of riddelliine metabolized by female rat liver microsomes. The chromatographic peak eluting at 35.7-39.2 min contained the recovered

Xia et al.

Figure 1. Reversed-phase HPLC analysis of riddelliine metabolites formed from metabolism of riddelliine by (A) female rat liver microsomes, (B) male rat liver microsomes, (C) female human liver microsomes, and (D) male human liver microsomes. HPLC analysis was conducted on a Prodigy 5 µm ODS column (10 × 250 mm, Phenomenex) eluted isocratically with 20 mM ammonium acetate buffer (pH 7) at 5 mL/min for 10 min followed with a linear gradient from 20 mM ammonium acetate buffer (pH 7.0) to 50% methanol in the buffer for 30 min.

substrate, riddelliine. On the basis of comparison of their HPLC retention times and UV-vis absorption spectra with those of synthetic standards and the metabolites of previous study (39), the materials contained in the chromatographic peaks eluting at 25.4 and 30.1 min were identified as DHP and riddelliine N-oxide, respectively. The HPLC profiles obtained from metabolism of riddelliine by male rat liver microsomes (Figure 1B), female human liver microsomes (F3) (Figure 1C), and male human liver microsomes (M2) (Figure 1D) were similarly analyzed. As shown in Figure 1, metabolism of riddelliine by male and female rat and human liver microsomes produced similar metabolism pattern. The chromatographic peaks eluted prior to 22 min were also found from incubation in the absence of riddelliine and, therefore, are presumably from microsomal proteins. In this study, metabolism of riddelliine by four male (samples M1-M4) and four female (samples F1-F4) human liver microsomes was conducted, and all the resulting metabolism patterns were similar. The rates of metabolite formation from all 10 rat and human liver microsomal incubations were determined. While the rat data are based on triplicate results, due to limit amount of human tissues available, human data are based on a single experiment (Table 1). The levels of DHP formation from female and male human liver microsomal metabolism were within a range of 0.20-0.62 nmol/min/mg protein. These levels are similar to or higher than those from male and female rat liver microsomal metabolism. The levels of riddelliine N-oxide formed from human liver microsomal incubations were in the range of 0.03-0.15 nmol/min/mg protein, which are similar to those from female rat liver microsomal metabolism, but significantly lower than those from male rat liver microsomal me-

Metabolism and DNA Adduct Formation of Riddelliine

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Table 1. Rates of Metabolic Formation of DHP and Reddelliine N-Oxide from Metabolism of Riddelliine by Liver Microsomes of Rat and Human rate of formation (nmol/min/mg protein) male rata

female rata

male humanb

female humanb

metabolite

MR

FR

M1

M2

M3

M4

F1

F2

F3

F4

DHP riddelliine N-oxide

0.21 ( 0.01 0.48 ( 0.02

0.14 ( 0.02 0.06 ( 0.01

0.31 0.06

0.62 0.15

0.49 0.11

0.32 0.05

0.29 0.06

0.20 0.03

0.27 0.05

0.50 0.12

a Data based on triplicate results and expressed as means ( SD. b Data based on a single experiment due to the limited amount of human tissues available.

Table 2. Inhibition Effect of Triacetyloleandomycin (TAO) of P450 3A4 on Riddelliine Metabolism by Human Liver Microsomes DHP liver microsomes

P450 3A4 inhibitor

male (M2) TAO female (F4) TAO

riddelliine N-oxide

nmol/min/mg protein

percentage of inhibition (%)

nmol/min/mg protein

percentage of inhibition (%)

1.50 ( 0.03 0.23 ( 0.02 1.00 ( 0.03 0.16 ( 0.01

85

0.36 ( 0.02 0.03 ( 0.004 0.25 ( 0.01 0.02 ( 0.002

92

84

92

Table 3. Enzyme Kinetic Parameters of Riddelliine Oxidative Metabolism in Rat and Human Liver Microsomesa kinetic parameters Vmax (nmol/min/mg protein) liver microsomes

DHP

riddelliine N-oxide

female ratb (FR) male ratc (MR) human, femaled human, male (M4)

0.48 ( 0.03 1.12 ( 0.04 1.70 ( 0.09 0.95 ( 0.02

0.30 ( 0.01 2.17 ( 0.08 0.43 ( 0.03 0.26 ( 0.01

Km (mM) DHP

riddelliine N-oxide

0.37 ( 0.05 0.28 ( 0.03 0.66 ( 0.08 0.24 ( 0.02

0.44 ( 0.04 0.25 ( 0.03 0.71 ( 0.12 0.44 ( 0.06

a Kinetic parameters, represented as means ( SD (n ) 3), were determined by employing GraphPad Prism software. b Liver microsomes were prepared by combining liver tissues of six female rats. c Liver microsomes were prepared by combining liver tissues of five male rats. d Combining equal amount of liver microsomal protein from the four female human liver microsomes samples (F1, F2, F3, and F4).

tabolism. Male rat liver microsomal metabolism is the only example where the rate of riddelliine N-oxide formation is higher than that of DHP (Table 1). The rates of formation of DHP vs riddelliine N-oxide from human liver microsomal metabolism were compared. A linear relationship exists between the rates of DHP formation and the rates of riddelliine N-oxide formation from the four female human liver microsomal metabolism, with a correlation coefficient of r2 > 0.99. A similar relationship was obtained from male human liver microsomal metabolism (r2 > 0.99). A linear relationship (r2 ) 0.99) was also obtained on the rates of formation from all the eight male and female human liver microsomal metabolism. Metabolism of Riddelliine by Human Liver Microsomes in the Presence of P450 3A4 Inhibitor Triacetyleandomycin (TAO). To determine whether P450 3A4 is the principal metabolizing enzyme that catalyzes metabolism of riddelliine to DHP and riddelliine N-oxide, metabolism of riddelliine by male (M2) and female (F4) human liver microsomes was conducted in the presence TAO, a specific P450 3A4 inhibitor. As shown in Table 2 and Figure 2, the DHP metabolite formed from the male and the female human microsomal metabolism was 85 and 84% reduced, respectively, and formation of riddelliine N-oxide was 92% inhibited in both male and female human microsomal metablism. Kinetic Parameters of Riddelliine Metabolism by Rat and Human Liver Microsomes. Kinetic parameters (Vmax and Km) of metabolism of riddelliine were determined. The determinations include those from metabolism with liver microsomes prepared by (i) pooling livers of six female rats, (ii) pooling livers of five male

Figure 2. Inhibition of human liver microsomal metabolism of riddelliine by the P450 3A4 inhibitor TAO. Male human liver microsomes M2 and female human liver microsomes F4 were used for this study. The HPLC analysis of the resulting DHP and riddelliine N-oxide metabolites was conducted under conditions as those in the legend of Figure 1.

rats, (iii) combining equal amount of liver microsomes from the four female human liver samples (F1, F2, F3, and F4), and (iv) one male human (M4) liver microsomes. The Vmax and Km values from male and female rat and human liver microsomal metabolism are summarized in Table 3. As an illustration, the substrate concentrationdependent initial velocity of DHP and riddelliine N-oxide formation from female rat liver microsomal metabolism is shown in Figure 3A. The Lineweaver-Burke doublereciprocal plots for DHP and riddelliine N-oxide are shown in Figure 3, panels B and C, respectively. These data indicate that DHP was formed at a higher Vmax rate

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Xia et al.

Figure 4. 32P-Postlabeling/HPLC analysis of DHP-derived DNA adducts formed from metabolism of riddelliine by liver microsomes of male rat liver microsomes, female rat liver microsomes, four individual male human liver microsomes, and four individual female human liver microsomes in the presence of calf thymus DNA. HPLC analysis was conducted on a Prodigy 5 µm ODS column (Phenomenex, 4.6 × 250 mm) and eluted isocratically with 20 mM sodium phosphate buffer (pH 4.5) for 10 min, followed by a 60-min linear gradient of 20 mM sodium phosphate buffer (pH 4.5) to 15% methanol in 20 mM sodium phosphate buffer. 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 was equipped with a diverter, and the eluent from the first 40 min was diverted from the radioactivity detector.

Figure 3. Female rat liver microsomal metabolism of riddelliine, (A) the substrate concentration-dependent initial velocity of DHP and riddelliine N-oxide formation, and the LineweaverBurke double-reciprocal plots for (B) DHP and (C) riddelliine N-oxide.

than riddelliine N-oxide. The results of female rat and human liver microsomal metabolism are compared. The Km values of the DHP formation are 0.37 ( 0.05 and 0.66 ( 0.08 mM from female rats and female humans, respectively (Table 3). The Vmax values of the female rat and human liver microsomal metabolism are 0.48 ( 0.03 and 1.70 ( 0.09 nmol/min/mg protein, respectively. It is necessary to note that because the metabolism is via multienzyme complex (microsomes rather than purified enzyme), the kinetics parameters addressed are apparent rather than absolute.

Human Liver Microsomal-Mediated DHP-Derived DNA Adduct Formation. To determine the relevance of our mechanistic study on rodents to humans, DHP-derived DNA adduct formation mediated by human liver microsomal metabolism of riddelliine was studied, and the results were compared with those from female F344 rat liver microsomal metabolism in the presence of calf thymus DNA. Incubation conditions are similar to those described above for metabolism of riddelliine by rat and human liver microsomes. The levels of DHPderived DNA adducts formed by female and male rat and human liver microsomal metabolism were analyzed by 32P-postlabeling/HPLC. As previously determined (39, 40), reaction of DHR and calf thymus DNA resulted in a set of eight DHP-derived DNA adducts (Figure 4A). These eight DHP-derived DNA adducts contained in the chromatographic peaks eluted at 47.6, 48.3, 51.4, 53.9, 55.3, 60.1, 61.0, and 62.6 min are 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 (40). Metabolism of riddelliine by female and male rat and human

Metabolism and DNA Adduct Formation of Riddelliine

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Figure 5. Formation of total DHP-derived DNA adducts from male F344 rat liver microsomes (MR), female F344 rat liver microsomes (FR), male human liver microsomes (M1, M2, M3, and M4), and female human liver microsomes (F1, F2, F3, and F4) in the presence of calf thymus DNA. Data represent the mean ( SD (n ) 3).

liver microsomes in the presence of calf thymus DNA also produced the same set of eight DHP-derived DNA adducts. The HPLC profiles resulting from liver microsomal metabolism of female rats, male rats, female human (F2), and male human (M4) are shown in Figure 4, panels B and E. The levels of the total DHP-derived DNA adducts formed from all the 10 male and female rat and human liver microsomes are shown in Figure 5. These results clearly indicate that the same set of eight DHP-derived DNA adducts was formed in all the cases. Furthermore, the quantities of DNA adducts formed from human microsomal metabolism are either similar to or 2-3-fold higher than those from rat liver microsomal metabolism.

Discussion In this study, we report that riddelliine is metabolized by male and female human liver microsomes to form DHP which leads to the formation of a set of eight DHPderived DNA adducts. The metabolism pattern and DNA adduct profiles from human liver microsomes are very similar to those formed in rat liver in vitro and in vivo (39). The kinetic parameters, Vmax and Km, from human liver microsomal metabolism are also comparable to those from rat liver microsomal metabolism. Taken together, these results strongly indicate that our previous in vivo and in vitro mechanistic studies with laboratory rodents (39) are highly relevant to humans. Since riddelliine induces liver tumors in male and female rats and male mice (24) and the DHP-derived DNA adducts are responsible for liver tumor induction, the results in the present study suggest that riddelliine can be highly genotoxic to humans and the genotoxic mechanism is mediated by the eight DHP-derived DNA adducts. The proposed metabolic activation of riddelliine leading to genotoxicity mediated by DNA adduct formation is shown in Figure 6. The metabolism study in the presence of TAO, a human P450 3A4 inhibitor, strongly suggests that the formations of DHP and riddelliine N-oxide from metabolism of riddelliine are both principally catalyzed by the P450 3A4 enzyme or by two or more closely related enzymes which under a combined condition produce similar results. It has been reported that hydroxylation of pyrrolizidine alkaloids at the necine base, particularly at the C-8 and C-3 positions to form 8- and 3-hydroxynecine derivatives, followed by dehydration to form the corresponding dehydropyrrolizidine (pyrrolic) derivatives, is a general metabolism pathway (2). Thus, we propose that metabolism of riddelliine catalyzed by P450 3A4

Figure 6. Proposed metabolic activation of riddelliine leading to DHP-derived DNA adduct formation in rodent and human liver microsomal metabolism.

enzyme first provides 8-hydroxyriddelliine and/or 3-hydroxyriddelliine as the primary metabolites, which upon enzymatic dehydration produced dehydroriddelliine (Figure 6). As previously discussed, there are two possible pathways that lead to DHP-derived DNA adducts. The first pathway is the covalent binding of dehydroriddelliine to cellular DNA followed by hydrolysis, either enzymatically or chemically, to the eight DHP-derived DNA adducts (Figure 6). The second pathway is that dehydroriddelliine is hydrolyzed, catalyzed by esterases and/or other hepatic enzymes, to form DHP, which subsequently binds to DNA. To date, it is not known which pathway is predominant in DHP-derived DNA adduct formation. It warrants further investigations. Our finding that the formation of DHP and riddelliine N-oxide from riddelliine are principally catalyzed by the P450 3A4 enzyme is consistent with that published by Miranda et al. (28) that P450 3A4 is the major metabolizing enzyme catalyzing the metabolism of senecionine, a tumorigenic pyrrolizidine alkaloid, to DHP and senecionine N-oxide in male and female human liver microsomes. However, the metabolizing enzymes in catalyzing these human metabolism are different from those in laboratory animals. For example, it has been shown that P450 2B was involved in the metabolism of a number of pyrrolizidine alkaloids to the DHP metabolite in guinea pig, hamsters, and sheep (33, 35, 37). It has also been determined that flavin-containing monooxygenase is involved in the conversion of pyrrolizidine alkaloids to the corresponding N-oxides (2, 38). Although a number of pyrrolizidine alkaloids have been found to induce tumors in laboratory animals (3, 4, 6, 39), the mechanisms of these compounds leading to tumorigenicity are not clear. Our previous study on riddelliine provides the first established genotoxic mechanism by which a pyrrolizidine alkaloid (riddelliine) induces liver tumors mediated by the formation of eight DHP-derived DNA adducts. We anticipate that this activation pathway may be general and the eight DHPderived DNA adducts may be formed in vivo and in vitro when exposure occurs to other genotoxic pyrrolizidine alkaloids. Since this present study has shown that the same genotoxic mechanism of metabolic activation of riddelliine exists in humans, further investigations are

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warranted as to whether human liver can metabolize other pyrrolizidine alkaloids through the same mechanism.

Acknowledgment. We thank Dr. Paul Howard for the provision of triacetyloleandomycin and Dr. Frederick A. Beland for critical review of this manuscript. This research was supported in part by an Interagency Agreement 224-93-0001 between the Food and Drug Administration/National Center for Toxicological Research (FDA/ NCTR) and the National Institute for Environmental Health Sciences/National Toxicology Program (NIEHS/ NTP). Through this agreement, this research was supported 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. Note Added after Print Publication: The copyright line was incorrect when this paper was published on the Web December 10, 2002 (ASAP) and in print [(2003) Chem. Res. Toxicol. 16, 66-73]. The electronic version was corrected on 2/13/2003 and an Addition and Correction appears in the March, 2003 issue (Vol. 16, No. 3).

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