Metabolic Activation of the Tumorigenic ... - ACS Publications

Riddelliine is a representative naturally occurring genotoxic pyrrolizidine alkaloid. We have studied the mechanism by which riddelliine induces hepat...
0 downloads 0 Views 99KB Size
Download PDF
Chem. Res. Toxicol. 2001, 14, 101-109

101

Metabolic Activation of the Tumorigenic Pyrrolizidine Alkaloid, Riddelliine, Leading to DNA Adduct Formation in Vivo Ya-Chen Yang,† Jian Yan,† Daniel R. Doerge,† Po-Cheun Chan,‡ Peter P. Fu,† and Ming W. Chou*,† National Center for Toxicological Research, Jefferson, Arkansas 72079, and National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 Received July 11, 2000

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-N2-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.

Introduction Riddelliine, a naturally occurring pyrrolizidine alkaloid (1) and a macrocyclic diester of retronecine and riddelliic acid (Scheme 1) (2, 3), is isolated from tansy ragwort (Senecio jacobaea) present in the rangelands in the western region of the United States. Serious economic loss to ranchers results from the poisoning of livestock grazing on plants containing riddelliine. Riddelliine may also contaminate human food sources, e.g., flowers or seeds in whole grain processed for flour, milk, and honey (2). The herbal tea named “gordolobo yerba”, popularly used in the American Southwest, may also contain riddelliine (4). Riddelliine exhibits both acute toxicity and genotoxic activity. It is mutagenic in Salmonella typhimurium TA100 in the presence of an S9 activation enzyme system (2, 5) and induces sister chromatid exchange and chromosomal aberrations in Chinese hamster ovary cells (6). These genotoxic activities are reduced when assayed in the absence of S9, suggesting that metabolism by enzymes present in the S9 system is required. Riddelliine induces unscheduled DNA synthesis in rat hepatocytes and in peripheral blood polychromatic erythrocytes of Swiss mice (7-9). It causes DNA cross-linking and DNAprotein cross-linking in cultured bovine kidney epithelial cells (10, 11) and shows a dose-dependent inhibition of colony formation and megalocytosis in the same cell line (12). In view of the human exposure to riddelliine, * To whom correspondence should be addressed. Telephone: (870) 543-7661. Fax: (870) 543-7719. E-mail: [email protected]. † National Center for Toxicological Research. ‡ National Institute of Environmental Health Sciences.

10.1021/tx000150n

Scheme 1. Chemical Structure and Numbering of Riddelliine

riddelliine has been a concern to the U.S. Food and Drug Administration (FDA)1 and was nominated by the FDA for genotoxicity and carcinogenicity testing conducted by the National Toxicology Program (NTP) (2, 3). Data from a 13 week NTP study of B6C3F1 mice and F344 rats treated with riddelliine indicated the induction of un1 Abbreviations: MN, micrococcal nuclease; SPD, spleen phosphodiesterase; DHR, dehydroretronecine [7-hydroxyl-1-(hydroxymethyl)-6,7dihydro-5H-pyrrolizine]; 3′-dGMP, 2′-deoxyguanosine 3′-monophosphate; 5′-dGMP, 2′-deoxyguanosine 5′-monophosphate; DHR-3′-dGMP adducts (I and II), 3′-monophosphate of 7-(deoxyguanosin-N2-yl)dehydrosupinidine; DHR-3′,5′-dG-bisphosphate adducts (I and II), 3′,5′-bisphosphate of 7-(deoxyguanosin-N2-yl)dehydrosupinidine; 3′dAMP, 2′-deoxyadenosine 3′-monophosphate; DHR-3′-dAMP adducts, 3′-monophosphate of 7-(deoxyadenosin-N6-yl)dehydrosupinidine; DHR3′,5′-dA-bisphosphate adducts, 3′,5′-bisphosphate of 7-(deoxyadenosinN6-yl)dehydrosupinidine; PNK, cloned T4 polynucleotide kinase; PB microsomes, liver microsomes of female F344 rats pretreated with phenobarbital; control microsomes, liver microsomes of untreated female F344 rats; NTP, National Toxicology Program; NCTR, National Center for Toxicological Research.

This article not subject to U.S. Copyright. Published 2001 by the American Chemical Society Published on Web 12/12/2000

102

Chem. Res. Toxicol., Vol. 14, No. 1, 2001

scheduled DNA synthesis and S-phase synthesis in primary hepatocytes (2, 3, 13). In mating trials in both species, pups from treated dams weighed less at birth and during suckling than control animals (2). Riddelliine also induced liver tumors in female rats (2, 3). Thus, the results indicate that riddelliine is genotoxic and carcinogenic in rodents. A 2 year chronic tumorigenicity bioassay of this compound has also been conducted by the NTP at the National Institute of Environmental Health Sciences (NIEHS), and the preliminary results indicated that riddelliine induced liver tumors in female F344 rats.2 However, the mechanisms by which riddelliine induces liver tumors in rodents are not known. Even though riddelliine and other pyrrolizidine alkaloids are considered genotoxic chemical carcinogens (14-17), no DNA adducts have been identified from any compounds of this class in vivo or in vitro. We have studied the mechanism by which riddelliine induces hepatocellular tumors in female F344 rats. The studies include metabolic activation of riddelliine, identification of activated metabolites, and employment of the newly developed 32P-postlabeling/HPLC methodlogy (described in the preceding paper) for identification and quantification of the riddelliine-derived DNA adducts in livers of riddelliine-treated female F344 rats.

Materials and Methods Materials. Riddelliine was obtained from the NTP. DHR and riddelliine N-oxide were prepared following published procedures (18-20). 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 Sigma Chemical Co. (St. Louis, MO). Cloned T4 polynucleotide kinase (PNK) was obtained from U.S. Biochemical Corp. (Cleveland, OH). Adenosine [γ-32P]-5′-triphosphate ([γ-32P]ATP) (specific activity of >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 were HPLC grade. The 3′-monophosphate of 7-(deoxyguanosin-N2-yl)dehydrosupinidine adducts (DHR-3′-dGMP) and the 3′-monophosphate of 7-(deoxyadenosin-N6-yl)dehydrosupinidine adducts (DHR-3′-dAMP) were prepared in our laboratory (described in the preceding paper). When measured in water, the molar extinction coefficient of DHR-3′-dGMP adducts at 254 nm was determined to be 4.7 × 104 M-1 cm-1 (described in the preceding paper). DNA from the reaction of DHR with calf thymus DNA was prepared as previously described (described in the preceding paper). Animals. Female F344 rats were obtained from the National Center for Toxicological Research (NCTR) breeding colony as weanlings. Liver microsomes of female F344 rats treated with phenobarbital (PB microsomes) were prepared according to published procedures (21). Phenobarbital (75 mg/kg of body weight/day, in 0.5 mL of H2O) was injected intraperitoneally into rats for 3 consecutive days. Twenty-four hours after the final injection, rats were sacrificed by CO2 inhalation. The livers were perfused with cold 1.15% KCl via the portal vein and immediately stored at -78 °C. The liver microsomes were prepared from the thawed livers by differential centrifugation methods (21) and stored at -78 °C prior to use. Liver microsomes of untreated female F344 rats (control microsomes) 2

NTP, unpublished data.

Yang et al. Table 1. Liver Samples of Riddelliine-Treated Female F344 Rats from the NTP/NIEHS Chronic Bioassay dose (mg/kg/day)

no. of rats per dose per dosing period 3 months

6 months

0 0.01 0.033 0.1 0.33 1.0

6 6 6 6 6 6

6 6 6 6 6 6

total

36

36

were prepared similarly. 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). A chronic animal study using a total of 72 female F344 rats, divided into 12 experimental groups (six rats per group), was conducted by the NTP. The rats in each group were orally gavaged (5 days/week) with vehicle (water) and 0.01, 0.033, 0.1, 0.33, and 1.0 mg of riddelliine/kg/day, respectively, beginning at weaning and continuing until sacrifice 3 or 6 months after the first treatment day (Table 1). After sacrifice, liver tissue was collected, stored at -78 °C, and shipped to the NCTR for DNA adduct analysis. Metabolism of Riddelliine. Metabolism of riddelliine by PB microsomes was performed 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, 4 mg of PB microsomes, and riddelliine (2 µmol in 50 µL of DMSO) at 37 °C for 30 min. The incubation mixture was centrifuged at 105000g for 30 min at 4 °C to remove microsomal proteins. The resulting supernatant was collected, and the resulting metabolite mixture was separated by reversed-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) at a flow rate of 1 mL/min for 10 min followed with a linear gradient from 20 mM ammonium acetate buffer (pH 7) to 50% methanol in the buffer for 40 min. The metabolite mixture was subsequently analyzed by LC/MS. Metabolism of riddelliine by control microsomes was similarly conducted, and the metabolites were analyzed by HPLC and LC/MS. Reaction of Riddelliine with Calf Thymus DNA. A DNA sample that served as a negative control was prepared by reaction of purified calf thymus DNA (2.5 mg, 7.5 µmol, dissolved in 2.5 mL of distilled water) with riddelliine (64 nmol) at 37 °C for 40 min. After incubation, the reaction mixture was extracted twice with 10 mL of a chloroform/isoamyl alcohol mixture (24/ 1, v/v). The DNA in the aqueous phase was precipitated by adding 250 µL of 3 M sodium acetate followed by equal volume of cold 2-propanol and washed with 70% ethanol. After the DNA had been dissolved in 20 mM K2CO3 (pH 7.5), the concentration and purity were analyzed spectrophotometrically. The DNA was stored at -78 °C prior to 32P-postlabeling/HPLC analysis. 32P-Postlabeling/HPLC Analysis of DHR-Derived (Riddelliine-Derived) DNA Adducts from Liver Samples Provided by the NTP. DHR-derived DNA adducts contained in the 72 female F344 rats (Table 1) provided by the NTP were analyzed according to recently developed 32P-postlabeling/HPLC methodology (described in the preceding paper). Briefly, 10 µg of the liver DNA, extracted by using the RecoverEase DNA Isolation Kit (Stratagene, Cedar Creek, TX) according to the manufacturer’s instructions, dissolved in 10 µL of distilled water was enzymatically hydrolyzed to the corresponding 2′-deoxyribonucleoside 3′-monophosphates 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 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 (pH 5)] at 37 °C for 20 min to

Metabolic Activation of Riddelliine remove the 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 mix containing 100 µCi of [γ-32P]ATP (specific activity of 7000 Ci/mmol), 12 units of PNK, and 2 µL of 10× PNK buffer [200 mM bicine-NaOH (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 (Phenomenex, 4.6 mm × 250 mm) 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 caused by the high level of 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. To identify the DHR-derived DNA adducts in the NTP liver samples, DNA from the reaction of DHR and calf thymus DNA was 32P-postlabeled and analyzed by HPLC in parallel. For quantitation of each sample, the two epimeric DHR-3′-dGMP synthetic standards in amounts that closely matched the range of modification in the liver DNA samples were also analyzed in parallel. DNA from the chemical reaction of riddelliine with calf thymus DNA was also subjected to 32P-postlabeling/HPLC analysis in parallel to serve as a negative control. The levels of adducts in vivo were compared by a two-way ANOVA combined with Dunnett’s test with dose and time as factors and total and individual adducts as dependent variables (Sigmastat, version 2.0, SPSS, Inc.). Separate analyses to compare individual adducts were performed by using the t test. Instrumentation. A Waters HPLC system consisted of a model 600 controller, a model 996 photodiode array detector, and a pump used for the separation and purification of DHRderived DNA adducts. Electrospray (ES) mass spectrometry was performed using a Platfoum II single-quadrupole instrument (Micromass, Inc., Altrincham, U.K.). ES tandem mass spectrometry was performed using a Quattro liquid chromatograph (Micromass). Separate MS functions were used to acquire full scan data at a low and high cone voltages in a single chromatographic run (e.g., 20 and 40 V, respectively, for m/z 100-600). ES tandem mass spectrometry was performed using the negative and/or positive ion mode with a source temperature of 80 °C for infusion with a syringe pump. Product ion scans were obtained from CID of selected ions using a cone voltage between 37 and 40 V and collision energies between 24 and 31 eV. The collision gas was Ar at pressures between 2 and 4 × 10-3 mbar. LC/MS samples (5 µL injection volume) were introduced into the ES probe following separation with a Prodigy 5 µm ODS column (Phenomenex, 4.6 mm × 250 mm), and eluting with the conditions previously described, and split to a rate of approximately 0.2 mL/min entering the probe. For radiochromatographic analysis of the 32P-postlabeled reaction mixtures, an HPLC system with on-line detection of both radioactivity and UV absorbance consisted of a solvent gradient programer (Waters model 680), two HPLC pumps (Waters model 510), a UV detector (Waters model 440), and a radiochromatography detector (Hewlett-Packard FLO-ONE /Beta A-500) equipped with a diverter and an autosampler (Waters model 717).

Results Metabolism of Riddelliine. Riddelliine was incubated aerobically with liver microsomes of rats pretreated with phenobarbital (PB microsomes) for 30 min. Upon removal of the microsomal protein by ultracentrifugation, the metabolism products contained in the supernatant were separated and analyzed by reversed-phase HPLC (Figure 1). The chromatographic peak eluting at 43-48

Chem. Res. Toxicol., Vol. 14, No. 1, 2001 103

Figure 1. Reversed-phase HPLC analysis of riddelliine metabolites formed from metabolism of riddelliine by PB microsomes. HPLC analysis was conducted on a Prodigy 5 µm ODS column eluted with a linear gradient from 20 mM sodium phosphate buffer (pH 5) to 15% methanol in buffer for 40 min at a flow rate of 1.0 mL/min.

Scheme 2. Metabolism of Riddelliine by Rat Liver Microsomes

min contained the recovered substrate, riddelliine. By comparison of HPLC retention times, UV/visible absorption, and mass spectral data (not shown) with those of the synthetically prepared standards, the metabolites contained in chromatographic peaks eluting at 28.3 and 35.2 min were identified as dehydroretronecine (DHR) and riddelliine N-oxide (Figure 1 and Scheme 2). Since the chromatographic peaks that eluted prior to 26 min were also detected from a control incubation to which riddelliine had not been added, they were not metabolites. Mass spectral analysis of the materials contained in these peaks (data not shown) confirmed that no metabolites were present. Metabolism of riddelliine by liver microsomes of untreated rats (control microsomes) was conducted under similar conditions. On the basis of comparison of the HPLC peak area and UV/visible absorption intensity of the riddelliine N-oxide and DHR metabolites, the rate of metabolism of riddelliine by PB microsomes was 3.5-fold higher than that by control microsomes (Table 2). 32P-Postlabeling/HPLC Profiles of DHR-Derived DNA Adduct Standards. To analyze DHR-derived DNA adducts in liver of rats fed riddelliine, two control 32Ppostlabeling/HPLC profiles of DHR-derived DNA adduct standards were first analyzed. These were obtained from 32P-postlabeling/HPLC of DNA contained in (i) a mixture of synthetically prepared DHR-3′-dGMP and DHR-3′-

104

Chem. Res. Toxicol., Vol. 14, No. 1, 2001

Table 2. In Vitro Metabolism of Riddelliine by Liver Microsomes from Female F344 Rats Untreated and Pretreated with Phenobarbitala nmol of metabolite formed (mg of microsomal protein)-1 (30 min)-1 microsomes control PB-induced PB/control ratio

dehydroretronecine

riddelliine N-oxide

8.3 ( 0.7 31.1 ( 1.5

14.7 ( 1.1 50.0 ( 1.7

3.8

3.4

Data represent the mean ( SD (n ) 3). For the experimental details, see Materials and Methods. a

dAMP adducts and (ii) modified DNA from the reaction of DHR with calf thymus DNA. A negative control was obtained from the 32P-postlabeling/HPLC analysis of DNA obtained from incubation of riddelliine with calf thymus DNA. These HPLC profiles are shown in panels A-C of Figure 2. As shown in Figure 2A, the HPLC retention times of DHR-3′,5′-dG-bisphosphate adducts I and II are 53.9 and 60.1 min, respectively. Like previous data (described in the preceding paper), 32P-postlabeling of adducts from reaction of DHR and calf thymus DNA

Figure 2. 32P-postlabeling/HPLC analysis of DHR-derived DNA adducts contained in (A) a mixture of synthetically prepared DHR-3′-dGMP and DHR-3′-dAMP adducts, (B) modified DNA from the reaction of DHR with calf thymus DNA, and (C) DNA obtained from the chemical reaction of riddelliine with calf thymus DNA and in livers of rats treated by oral gavage with (D) vehicle (water) or with riddelliine at doses of 0.01 (E), 0.033 (F), 0.1 (G), 0.33 (H), and 1.0 mg/kg/day (I) for 5 days/week beginning at weaning and continuing until sacrifice at three months. The eight chromatographic peaks that eluted at 47.6, 48.3, 51.4, 53.9, 55.3, 60.1, 61.0, and 62.6 min are designated as P1-P8, respectively. The DNA adducts designated as P4 and P6 are DHR-3′-dGMP adduct I and adduct II, respectively. For the HPLC conditions, see Materials and Methods.

Yang et al.

resulted in eight DHR-derived DNA adducts (Figure 2B), which were not formed from the control experiment, incubation of riddelliine with calf thymus DNA (Figure 2C). These eight DHR-derived DNA adducts contained in the chromatographic peaks that eluted at 47.6, 48.3, 51.4, 53.9, 55.3, 60.1, 61.0, and 62.6 min are designated as P1P8, respectively. The DNA adducts designated as P4 and P6 are DHR-3′-dGMP adduct I and adduct II, respectively. Due to lack of synthetic standards, the structures of the other six DHR-derived adducts (P1-P3, P5, P7, and P8) were not characterized. 32 P-Postlabeling/HPLC Analysis of RiddelliineModified DNA Adducts in Liver Samples. The levels of riddelliine-modified DNA adducts contained in the DNA of the female rat liver samples (Table 1) were analyzed by 32P-postlabeling/HPLC analysis. It has been previously established that the 32P-postlabeling methodology has a high intraexperimental reproducibility, but the interexperimental reproducibility was much less satisfactory (described in the preceding paper). Thus, to conduct the experiment on the same day, four liver DNA samples from each of six groups were analyzed simultaneously. For identification and quantification of the modified 3′,5′-bisphosphate adduct, external standards were also analyzed in parallel with the biological samples. The standards are DHR-3′-dGMP adducts containing a known level of adducts (8.3 fmol, equal to 9.6 adducts/ 108 nucleotides) that corresponded closely to the modification level expected in each DNA sample. To verify the two chromatographic peaks of the [32P]DHR-3′,5′-dGbisphosphate adduct, an unlabeled synthetic standard of the DHR-3′,5′-dG-bisphosphate adduct was cochromatographed as an ultraviolet marker with the first two DNA samples from the liver samples. Verification of the chromatographic peaks of the other six DHR-derived DNA adducts was based on comparison of their HPLC retention times with those from DHR-modified calf thymus DNA (Figure 2B). The lack of [32P]DHR-3′,5′dA-bisphosphate adducts was based on comparison of HPLC retention times with those of the synthetic DHR3′,5′-dA-bisphosphate samples (Figure 2A). The DNA samples from liver of untreated rats and rats fed 0.01, 0.033, 0.1, 0.33, and 1.0 mg of riddelliine/kg/ day for 5 days/week beginning at weaning and continuing until sacrifice at 3 months were 32P-postlabeled and analyzed by HPLC (Figure 2D-I). The HPLC profiles of these samples (Figure 2D-I) are identical to that from the reaction of DHR and calf thymus DNA, albeit with different adduct amounts (Figure 2B). This comparison confirms that in the livers of rats fed riddelliine (i) all eight DHR-derived DNA adducts (P1-P8) were formed; (ii) among these eight DNA adducts, two were the epimeric [32P]DHR-3′,5′-dG-bisphosphate adducts I and II; and (iii) no DHR-3′,5′-dA-bisphosphate adducts were detected. As shown in Figure 2D, these eight DHRderived DNA adducts were not detected in the livers of untreated rats. Since the DHR-3′-dGMP standard containing 8.3 fmol (equal to 9.6 adducts/108 nucleotides) was 32P-postlabeled in parallel, the quantities of the two [32P]DHR-3′,5′-dGbisphosphate adducts I and II (P4 and P6, respecticely) in each of the liver samples were determined on the basis of comparison of the amount of radioactivity contained in the chromatographic peaks and in the peaks from the synthetic DHR-3′-dGMP standards. However, since the

Metabolic Activation of Riddelliine

Chem. Res. Toxicol., Vol. 14, No. 1, 2001 105

Table 3. 32P-Postlabeling/HPLC Quantification of Riddelliine-Derived DNA Adducts in Liver DNA of Female F344 Rats Fed Riddelliine for 3 Months in NTP Chronic Studiesa no. of adducts/108 nucleotides

dose (mg/kg/day)

P1 and P2

P3

P4 (adduct I)

P5

P6 (adduct II)

P7

P8

total

0 0.01 0.033 0.1 0.33 1.0b

0 20.0 ( 8.2 23.3 ( 5.3 47.6 ( 4.2 134.5 ( 28.0 199.6 ( 30.7

0 24.4 ( 15.4 33.5 ( 2.6 57.2 ( 3.0 128.2 ( 26.4 186.6 ( 14.6

0 2.6 ( 1.0 2.8 ( 0.1 3.9 ( 0.3 5.8 ( 0.2 8.3 ( 1.0

0 6.9 ( 2.5 8.0 ( 0.9 11.2 ( 0.7 42.4 ( 18.5 64.0 ( 7.8

0 0.9 ( 0.3 1.0 ( 0.2 1.4 ( 0.1 2.3 ( 0.4 3.2 ( 0.3

0 3.0 ( 0.9 3.6 ( 0.1 5.1 ( 0.2 8.6 ( 1.1 10.7 ( 2.3

0 1.8 ( 0.7 2.0 ( 0.1 2.8 ( 0.2 4.2 ( 0.5 5.9 ( 1.0

0 59.7 ( 28.9 74.1 ( 8.6 120.2 ( 8.7 326.0 ( 73.8 478.2 ( 48.5

a Animal dosing, liver DNA sample preparations, and 32P-postlabeling/HPLC analysis are described in Materials and Methods. All the liver DNA samples and triplicate DHR-dGMP adducts I and II were assessed under the same conditions and at the same time. P1-P8 represent the chromatographic fraction from 32P-postlabeling/HPLC assays. The retention times of P4 and P6 were identical to those of adducts I and adduct II, respectively. Data expreseed as the number of adducts per 108 nucleotides were calculated from the disintegrations per minute (dpm) of 32P radioactivities based on the results that 30 fmol of DHR-3′-dGMP adducts ) 28 480 dpm. Data represent means ( SD (n ) 4). Values of each individual adduct peak of all the dosing groups differ significantly from the control group (p < 0.01). The total adducts of samples from the 0.1, 0.33, and 1.0 dosing groups differ significantly from the control (0 dose) group (p < 0.05). b n ) 3 in this group.

Table 4. 32P-Postlabeling/HPLC Quantification of Riddelliine-Derived DNA Adducts in Liver DNA of Female F344 Rats Fed Riddelliine for 6 Months in NTP Chronic Studiesa no. of adducts/108 nucleotides

dose (mg/kg/day)

P1 and P2

P3

P4 (adduct I)

P5

P6 (adduct II)

P7

P8

total

0 0.01 0.033 0.1 0.33 1.0b

0 89.0 ( 37.4 157.7 ( 62.9 237.7 ( 58.8 411.2 ( 89.3 509.6 ( 43.1

0 155.6 ( 40.2 246.3 ( 53.4 353.6 ( 48.7 519.3 ( 41.8 720.7 ( 9.8

0 5.5 ( 1.0 7.4 ( 1.0 13.0 ( 6.7 22.4 ( 2.4 38.2 ( 2.3

0 56.5 ( 20.7 99.3 ( 35.3 141.0 ( 31.8 226.8 ( 27.6 300.8 ( 4.6

0 2.0 ( 0.4 3.5 ( 1.2 4.4 ( 0.2 7.0 ( 0.7 9.75 ( 1.0

0 9.2 ( 2.1 22.3 ( 5.8 37.0 ( 5.2 55.6 ( 3.1 85.5 ( 6.2

0 6.5 ( 0.6 10.3 ( 1.0 23.6 ( 4.1 38.0 ( 3.3 59.8 ( 7.1

0 324.3 ( 102.0 546.9 ( 158.6 810.2 ( 154.8 1280.3 ( 158.5 1724.3 ( 37.3

a Animal dosing, liver DNA sample preparations, and 32P-postlabeling/HPLC analysis are described in Materials and Methods. All the liver DNA samples and triplicate DHR-dGMP adducts I and II were assessed under the same conditions and at the same time. P1-P8 represent the chromatographic fraction from 32P-postlabeling/HPLC assays. The retention times of P4 and P6 were identical to those of adducts I and adduct II, respectively. Data expreseed as the number of adducts per 108 nucleotides were calculated from the dpm of 32P radioactivities based on the results that 30 fmol of DHR-dGMP adducts ) 28 480 dpm. Data represent means ( SD (n ) 4). Values of each individual adduct peak of all the dosing groups differ significantly from the control group (p < 0.01). The total adducts of samples from the 0.1, 0.33, and 1.0 dosing groups differ significantly from the control (0 dose) group (p < 0.05). b n ) 3 in this group.

structures of the other six DNA adducts (P1-P3, P5, P7, and P8) are not known and therefore no synthetic standards with known levels of modification can be used for quantification, the quantities of these six DNA adducts were estimated, by comparison of their amounts of radioactivity contained in the chromatographic peak and that of a known quantity (8.3 fmol) of the DHR-3′dGMP adduct synthetic standard (Table 3). The total quantities of DHR-derived DNA adducts that includes these six adducts and the two DHR-3′-dGMP adducts I and II (which are also designated as P4 and P6, respectively) are listed in Table 3. The DNA samples from liver of untreated rats and rats fed riddelliine and sacrificed at 6 months were similarly 32P-postlabeled, followed by HPLC analysis. The HPLC profile (data not shown) is closely similar to that of rats sacrificed at 3 months. The quantities of each adduct peak are listed in Table 4. The data shown in Table 3 indicate that a positive dose-response trend exists for DHR-3′-dGMP adducts I and II from the liver of rats fed riddelliine for 3 months (Figure 3). Similarly, the data listed in Table 4 also indicate a positive dose-response trend for adducts I and II from the liver of rats fed riddelliine for 6 months. The p values for adducts I and II are 0.009 and 0.012, respectively. Statistical analysis of the data shown in Tables 3 and 4 by a two-way ANOVA combined with a Dunnett’s test indicates that (i) compared with the animals fed the same dose of riddelliine, the levels of DNA adducts formed in the liver of rats fed riddelliine for 6 months are higher

Figure 3. Dose-response relationship of formation of DHR3′-dGMP adducts I and II contained in liver DNA of female 344 rats fed riddelliine for 3 and 6 months.

than those in rats fed for 3 months (p < 0.05); and (ii) in all the liver DNA from the riddelliine-treated rats, the yield of adduct I was higher than that of adduct II (p < 0.05). The data shown in Figure 4 indicate that a positive dose-response trend exists for the total DHR-derived DNA adducts from the liver of rats fed riddelliine for 3 and 6 months. Statistical analysis of the total amount of the DNA adducts formed by a two-way ANOVA combined with a Dunnett test indicates that (i) the total level of adduct formation of the 6 month group was greater than that of the 3 month group and (ii) all the groups, except

106

Chem. Res. Toxicol., Vol. 14, No. 1, 2001

the first two (0.01 and 0.033 mg/kg/day), of rats fed riddelliine for 3 months were significantly different from the control animals (p < 0.05). Confirmation of Optimal 32P-Postlabeling Conditions. It was previously observed that the digestion enzymes, MN and SPD, may interact with DHR-3′dGMP and/or [32P]DHR-3′,5′-dG-bisphosphate adducts (described in the preceding paper). To verify that optimal conditions were used for 32P-postlabeling, analyses were repeated using different amounts of the digestion enzymes. Thus, digestion of liver DNA (10 µg) of rats fed riddelliine was repeated by using (i) 1250 mU of MN and 62 mU of SPD, (ii) 312 mU of MN and 16 mU of SPD, (iii) 156 mU of MN and 8 mU of SPD, (iv) 78 mU of MN and 4 mU of SPD, and (v) 39 mU of MN and 2 mU of SPD. These results clearly confirm that the use of 78 mU of MN and 4 mU of SPD provided the highest yield of DHR-3′,5′-dG-bisphosphate adducts (data not shown).

Discussion Although pyrrolizidine alkaloids are a class of genotoxic naturally occurring phytochemicals and a number of pyrrolizidine alkaloids have been found to induce tumors in experimental animals (15, 17, 22-36), the mechanisms that lead to tumorigenicity are not clear. We report in this paper the detection and quantification of eight DHR-derived DNA adducts in livers of rats fed riddelliine by 32P-postlabeling/HPLC analysis. The liver samples are from female F344 rats fed riddelliine at five different doses and sacrificed after being maintained on these doses for two different lengths of time (Table 1). A dose-response relationship was obtained between the dose and level of total DNA adducts, which are all derived from DHR (Tables 3 and 4). The DNA from the reaction of riddelliine and calf thymus DNA was similarly analyzed by the 32P-postlabeling/HPLC method. In this case,

Yang et al.

Figure 4. Dose-response relationship of total riddelliinederived DNA adduct formation in liver DNA of rats fed riddelliine for 3 and 6 months.

no DHR-derived DNA adducts were formed (Figure 2C), indicating that riddelliine cannot bind with DNA in the absence of metabolism. We have also demonstrated that metabolism of riddelliine by rat liver microsomes formed DHR as a major metabolite (Table 2). Thus, these combined results suggest that riddelliine induces liver tumors in rats through a genotoxic mechanism. Eight DHR-derived DNA adducts were detected in all the liver samples of rats treated with riddelliine, among which two adducts had their structures fully characterized as epimeric DHR-3′-dGMP adducts (see Figure 2 and the preceding paper). As shown in Tables 3 and 4, the total quantity of the two DHR-3′-dGMP adducts, accounting for 2-6% of the total extent of DNA adduct formation, was much lower than the other six unidentified adducts. Since a dose relationship exists, these eight DHR-derived DNA adducts may be responsible in part, if not solely,

Scheme 3. Proposed Metabolic Activation of Riddelliine Leading to DHR-Modified DNA Adducts and Liver Tumor Formation in Female F344 Rats Fed Riddelliine

Metabolic Activation of Riddelliine

for development of liver tumors in the female rats fed riddelliine. In our study, microsomal metabolism of riddelliine generated riddelliine N-oxide and DHR. The formation of these metabolites is also in agreement with the in vitro metabolism pattern reported for other pyrrolizidine alkaloids (17, 37-44). It has been well established that both P450 2B6 and 3A4 isozymes are the major metabolizing enzymes responsible for pyrrolizidine alkaloid metabolism (39, 41, 45-48). In our study, we have also found that the rate of riddelliine metabolism was higher with PB microsomes than with control microsomes. Since phenobarbital induces both P450 2B6 and 3A4 enzymes (49), these results suggest that P450 2B6 and/or 3A4 enzymes are the major metabolizing enzymes responsible for riddelliine metabolism and that our results are consistent with those reported in the literature (39, 41, 45-48). On the basis of these results, the proposed metabolic activation of riddelliine leading to liver tumor formation in rats is shown in Scheme 3. 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, respectively, followed by dehydration to form the corresponding dehydropyrrolizidine (pyrrolic) derivatives, is a general metabolism pathway (14, 17). Thus, we propose that metabolism of riddelliine catalyzed by P450 enzymes first provides 8-hydroxyriddelliine and/or 3-hydroxyriddelliine as the primary metabolites, which upon enzymatic dehydration produced the dehydroriddelliine (Scheme 3). There are two possible pathways that lead to DHR-derived DNA adducts. The first pathway is that dehydroriddelliine, a potent electrophile, covalently binds to cellular DNA to form dehydroriddelliine-derived DNA adducts, which are subsequently hydrolyzed to DHR-derived DNA adducts. The second pathway is that dehydroriddelliine, like the other dehydropyrrolizidine alkaloids, is unstable and easily hydrolyzed by esterases and/or other hepatic enzymes to form DHR (15, 17, 50, 51), which subsequently binds to DNA. At present, it is not known from which pathway these eight DHR-derived DNA adducts are formed. Since dehydropyrrolizidine alkaloids (pyrroles) are highly unstable and DHR is the most stable pyrrolic compound (6, 52), more binding should occur through DHR rather than through dehydroriddelliine. 32 P-postlabeling methodology is a highly sensitive method for detecting and quantifying carcinogen-modified DNA adducts in vivo and in vitro (53-67). In our study, the postlabeling products, DHR-3′,5′-dG-bisphosphate adducts, were characterized by mass spectrometry and confirmed by cochromatography with synthetic DHR3′,5′-dG-bisphosphate standards. In each experiment, external standards, a pair of epimeric DHR-3′-dGMP synthetic adducts at a known level that closely matched the range of the modification level of the biological DNA samples, were also analyzed in parallel with the biological samples. Thus, this 32P-postlabeling/HPLC methodology enabled precise identification and quantification of the eight DHR-derived DNA adducts present in the liver of rats fed riddelliine.

Acknowledgment. We thank Jasyl Nichols for assisting in animal experiments and Dr. Frederick A. Beland for critical review of the manuscript. This research was supported in part by Interagency Agreement 224-93-0001 between the Food and Drug Administration/

Chem. Res. Toxicol., Vol. 14, No. 1, 2001 107

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 appointments (Y.-C.Y. and J.Y.) 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) Roeder, E. (1995) Medicinal plants in Europe containing pyrrolizidine alkaloids. Pharmazie 50, 83-98. (2) Chan, P. C., Mahler, J., Bucher, J. R., Travlos, G. S., and Reid, J. B. (1994) Toxicity and carcinogenicity of riddelline following 13 weeks of treatment to rats and mice. Toxicon 32, 891-908. (3) Chan, P. C. (1993) NTP Technical Report on Toxicity Studies of Riddelline (CAS No. 23246-96-0) Administered by Gavage to F344/N Rats and B6C3F1 Mice, NIH Publication 94-3350, U.S. Government Printing Office, Washington, DC. (4) Huxtable, R. J. (1980) Herbal teas and toxins: novel aspects of pyrrolizidine poisoning in the United States. Perspect. Biol. Med. 24, 1-14. (5) Zeiger, E., Anderson, B., Haworth, S., Lawlor, T., and Mortelmans, K. (1988) Salmonella mutagenicity tests. IV. Results from the testing of 300 chemicals. Environ. Mol. Mutagen. 11 (Suppl. 12), 1-158. (6) Galloways, S., Armstrong, M., Reuben, C., Colman, S., Brown, B., Cannon, C., Bloom, A., Namamura, F., Ahmed, M., Muk, S., Rimpo, J., Margolin, B., Resnick, M., Anderson, B., and Zeiger, E. (1987) Chromosome aberrations and sister chromatid exchanges in Chinese hamster ovary cells: evaluation of 108 chemicals. Environ. Mol. Mutagen. 10 (Suppl. 10), 1-175. (7) Mirsalis, J., Tyson, K., Beck, J., Loh, E., Steinmetz, K., Contreras, C., Auster, L., Markin, S., and Spalding, J. (1983) Induction of unscheduled DNA synthesis (UDS) in hepatocytes following in vitro and in vivo treatment. Environ. Mutagen. 5, 482 (abstract). (8) Mirsalis, J. (1987) In vivo measurement of unscheduled DNA synthesis and S-phase synthesis as an indicator of hepatocarcinogenesis in rodents. Cell Biol. Toxicol. 3, 165-173. (9) MacGregor, J., Hennika, P., and Roitman, J. (1985) Induction of micronuclei in peripheral blood erythrocytes of adult and fetal Swiss mice by pyrrolizidine alkaloids. Environ. Mutagen. 7 (Suppl. 3), 68. (10) Hincks, J. R., Kim, H.-Y., Segall, H. J., Molyneux, R. J., Stermitz, F. R., and Coulombe, R. A., Jr. (1991) DNA cross-linking in mammalian cells by pyrrolizidine alkaloids: structure-activity relationships. Toxicol. Appl. Pharmacol. 111, 90-98. (11) Kim, H. Y., Stermitz, F. R., and Coulombe, R. A., Jr. (1995) Pyrrolizidine alkaloid-induced DNA-protein cross-links. Carcinogenesis 16, 2691-2697. (12) Kim, H.-Y., Stermitz, F. R., Molyneux, R. J., Wilson, D. W., Taylor, D., and Coulombe, R. A., Jr. (1993) Structural influences on pyrrolizidine alkaloid-induced cytopathology. Toxicol. Appl. Pharmacol. 122, 61-69. (13) Mirsalis, J. C., Steinmetz, K. L., Blazak, W. F., and Spalding, J. W. (1993) Evaluation of the potential of riddelliine to induce unscheduled DNA synthesis, S-phase synthesis, or micronuclei following in vivo treatment with multiple doses. Environ. Mol. Mutagen. 21, 265-271. (14) Bull, L. B., Culvenor, C. C. J., and Dick, A. J. (1968) The Pyrrolizidine Alkaloids. Their Chemistry, Pathogenicity and other Biological Properties, North-Holland, Amsterdam. (15) International Agency for Research on Cancer (1976) Pyrrolizidine alkaloids. In IARC Monograph on the Evaluation of Carcinogenic Risk of Chemicals to Man: Some Naturally Occurring Substance, Vol. 10, pp 265-342, International Agency for Research on Cancer, Lyon, France. (16) World Health Organization (1988) Pyrrolizidine Alkaloids. In International Programme on Chemical Safety, Environmental Health Criteria, Vol. 80, p 337, World Health Organization, Geneva. (17) Mattocks, A. R. (1986) Chemistry and Toxicology of Pyrrolizidine Alkaloids, Academic Press, New York. (18) Hoskins, W. M., and Crout, D. H. G. (1977) Pyrrolizidine alkaloid analogues. Preparation of semisynthetic esters of retronecine. J. Chem. Soc., Perkin Trans 1, 538-543. (19) Mattocks, A. R., Jukes, R., and Brown, J. (1989) Simple procedures for preparing putative toxic metabolites of pyrrolizidine alkaloids. Toxicon 27, 561-569.

108

Chem. Res. Toxicol., Vol. 14, No. 1, 2001

(20) Christie, M. H., Kropman, M., Leisegang, E. C., and Warren, F. L. (1949) The Senecio Alkaloids. Part III. The structure of retrorsine and isatidine, and the isomerism of retronecic acid and isatinecic acid. J. Chem. Soc., 1700-1702. (21) 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. (22) Schoental, R. (1976) Carcinogens in plants and microorganisms. In Chemical Carcinogens (Searle, C. E., Ed.) ACS Monograph 173, American Chemical Society, Washington, DC. (23) Schoental, R., Head, M. A., and Peacock, P. R. (1954) Senecio alkaloids: primary liver tumors in rats as a result of treatment with (i) a mixture of alkaloids from S. jacobaea linn., (ii) retrorsine, (iii) isatidine. Br. J. Cancer 8, 458-465. (24) Newbern, P. N., and Rogers, A. E. (1973) Nutrition, monocrotaline and aflatoxin B1 in liver carcinogenesis. Plant Foods Man, 2331. (25) Svoboda, D. J., and Reddy, J. K. (1972) Malignant tumors in rats given lasicarpine. Cancer Res. 32, 908-913. (26) Schoental, R., and Head, M. (1957) Progression of liver lesions produced in rats by temporary treatment with pyrrolizidine (senecio) alkaloids, and the effects of betaine and high casein diet. Br. J. Cancer 11, 535-544. (27) 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. (28) 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. (29) Harris, P. N., and Chen, K. K. (1970) Development of hepatic tumors in rats following ingestion of Senecio longilobus. Cancer Res. 30, 2881-2886. (30) 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. (31) Schoental, R., Fowler, M. E., and Coady, A. (1970) Islet cell tumors of the pancreas found in rats given pyrrolizidiine alkaliods from Amsinckia intermedia Fisch and Mey and from Heliotropium supinum L. Cancer Res. 30, 2127-2131. (32) 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. (33) Schoental, R. (1975) Pancreatic islet-cell and other tumors in rats given heliotrine, a monoester pyrrolizidine alkaloid, and nicotinamide. Cancer Res. 35, 2020-2024. (34) Rao, M. S., and Reddy, J. K. (1978) Malignant neoplasms in rats fed lasiocarpine. Br. J. Cancer 37, 289-293. (35) Mattocks, A. R., and Cabral, J. R. (1982) Carcinogenicity of some pyrrolic pyrrolizidine alkaloid metabolites and analogues. Cancer Lett. 17, 61-66. (36) Cook, J. W., Duffy, E., and Schoental, R. (1950) Primary liver tumors in rats following feeding with alkaloids of Senecio jacobaea. Br. J. Cancer 4, 405-412. (37) Eastman, D. F., Dimenna, G. P., and Segall, H. J. (1982) Covalent binding of two pyrrolizidine alkaloids, senecionine and seneciphylline, to hepatic macromolecules and their distribution, excretion, and transfer into milk of lactating mice. Drug Metab. Dispos. 10, 236-240. (38) Mattocks, A. R., and Bird, I. (1983) Alkylation by dehydroretronecine, a cytotoxic metabolite of some pyrrolizidine alkaloids: an in vitro test. Toxicol. Lett. 16, 1-8. (39) Williams, D. E., Reed, R. L., Kedzierski, B., Dannan, G. A., Guengerich, F. P., and Buhler, D. (1989) Bioactivation and detoxication of the pyrrolizidine alkaloid senecionine by cytochrome P-450 enzymes in rat liver. Drug Metab. Dispos. 17, 387392. (40) Huxtable, R. J., and Wild, S. L. (1994) Relationship between in vitro metabolism of pyrrolizidine alkaloids and extrahepatic toxicity in vivo. Proc. West. Pharmacol. Soc. 37, 109-111. (41) Chu, P. S., Lame, M. W., and Segall, H. J. (1993) In vivo metabolism of retrosine and retrosine-N-oxide. Arch. Toxicol. 67, 39-43. (42) Mattocks, A. R., and White, I. N. (1971) The conversion of pyrrolizidine alkaloids to N-oxides and to dihydropyrrolizine derivatives by rat-liver microsomes in vitro. Chem.-Biol. Interact. 3 (5), 383-396. (43) Segall, H. J., Dallas, J. L., and Haddon, W. F. (1984) Two dihydropyrrolizidine alkaloid metabolites isolated from mouse hepatic microsomes in vitro. Drug Metab. Dispos. 12, 68-71.

Yang et al. (44) White, I. N. H., Mattocks, A. R., and Butler, W. H. (1973) The conversion of the pyrrolizidine alkaloid retrorsine to pyrrolic derivatives in vivo and in vitro and its acute toxicity to various animal species. Chem.-Biol. Interact. 6, 207-218. (45) Reid, M. J., Lame, M. W., Morin, D., Wilson, D. W., and Segall, H. J. (1998) Involvement of cytochrome P450 3A in the metabolism and covalent binding of 14C-monocrotaline in rat liver microsomes. J. Biochem. Mol. Toxicol. 12, 157-166. (46) Kasahara, Y., Kiyatake, K., Tatsumi, K., Sugito, K., Kakusaka, I., Yamagata, S.-I., Ohmori, S., Kitada, M., and Kuriyama, T. (1997) Bioactivation of monocrotaline by P450 3A in rat liver. J. Cardiovasc. Pharmacol. 30, 124-129. (47) Chung, W.-G., Miranda, C. L., and Buhler, D. R. (1995) A cytochrome P2502B form is the major bioactivation enzyme for the pyrrolizidine alkaloid senecionine in guinea pig. Xenobiotica 25, 929-939. (48) Buhler, D. R., and Kedzierski, B. (1986) Biological reactive intermediates of pyrrolizidine alkaloids. Adv. Exp. Med. Biol. 197, 611-620. (49) Omiecinski, C., Remmel, R. P., and Hosagrahara, V. P. (1999) Concise review of the cytochrome P450s and their roles in toxicity. Toxicol. Sci. 48, 151-156. (50) Mattocks, A. R. (1968) Toxicology of pyrrolizidine alkaloids. Nature 217, 723-728. (51) Kim, H. Y., Stermitz, F. R., Li, K.-K. J., and Coulombe, R. A., Jr. (1999) Comparative DNA cross-linking by activated pyrrolizidine alkaloids. Food Chem. Toxicol. 37, 619-625. (52) Huxtable, R J., Yan, C. C., Wild, S., Maxwell, S., and Cooper, R. (1996) Physicochemical and metabolic basis for the differing neurotoxicity of the pyrrolizidine alkaloids, trichodesmine and monocrotaline. Neurochem. Res. 21, 141-146. (53) Reddy, M. V., and Randerath, K. (1986) Nuclease P1-mediated enhancement of sensitivity of 32P-postlabelling test for structurally diverse DNA adducts. Carcinogenesis 7, 1543-1551. (54) Gupta, R. C. (1993) 32P-Postlabelling analysis of bulky aromatic adducts. In Postlabelling Methods for Detection of DNA Adducts (Phillips, D. P., Castegnaro, M., and Bartsch, H., Eds.) pp 1123, International Agency for Research on Cancer, Lyon, France. (55) Nath, R. G., and Chung, F.-L. (1994) Detection of exocyclic 1,N2propanodeoxyguanosine adducts as common DNA lesions in rodents and humans. Proc. Natl. Acad. Sci. U.S.A. 91, 7491-7495. (56) Chung, F.-L., Chen, H.-Y. C., and Nath, R. G. (1996) Lipid peroxidation as a potential endogenous source for the formation of exocyclic DNA adducts. Carcinogenesis 17, 2105-2111. (57) Zhan, D.-J., Herreno-Saenz, D., Chiu, L.-H., Von Tungeln, L. S., Wu, Y.-S., Lewtas, J., and Fu, P. P. (1995) Separation of 32P-labeled 3′,5′-bisphosphate nucleotides of polycyclic aromatic hydrocarbon anti-diol-epoxides and derivatives. J. Chromatogr., A 710, 149-157. (58) Herreno-Saenz, D., Evans, F. E., Beland, F. A., and Fu, P. P. (1995) Identification of two N2-deoxyguanosinyl DNA adducts upon nitroreduction of the environmental mutagen 1-nitropyrene. Chem. Res. Toxicol. 8, 269-277. (59) Herreno-Saenz, D., Evans, F. E., Lai, C. C., Fu, P. P., and Delclos, K. B. (1993) Characterization of the products of the in vitro reaction of metabolites of 3-aminochrysene with DNA. Chem.Biol. Interact. 86, 1-15. (60) Herreno-Saenz, D., Von Tungeln, L. S., Heflich, R. H., Lewtas, J., and Fu, P. P. (1994) Formation of N-(deoxyguanosin-8-yl)-2aminofluoranthene as a major 2-nitrofluoranthene induced DNA adduct in Salmonella typhimurium and neonatal B6C3F1 mice. Polycyclic Aromat. Compd. 6, 79-85. (61) Herreno-Saenz, D., Evans, F. E., and Fu, P. P. (1994) Nitroreduction of 1- and 3-nitro-7,8,9,10-tetrahydrobenzopyrene resulting in the N2-deoxyguanosinyl adducts through long-range migration. Chem. Res. Toxicol. 7, 806-814. (62) Ni, Y.-C., Kadlubar, F. F., and Fu, P. P. (1995) Formation of malondialdehyde-modified 2′-deoxyguanosinyl adduct from metabolism of chloral hydrate by mouse liver microsomes. Biochem. Biophys. Res. Commun. 216, 1110-1117. (63) Fu, P. P., Zhan, D.-J., Von Tungeln, L. S., Yi, P., Qui, F.-Y., Herreno-Saenz, D., and Lewtas, J. (1996) Comparative formation of DNA adducts of nitro-polycyclic aromatic hydrocarbons in mouse and rat liver microsomes and cytosols. Polycyclic Aromat. Compd. 10, 187-194. (64) Fu, P. P., Von Tungeln, L. S., Zhan, D.-J., and Bucci, T. (1996) Potent tumorigenicity of 7-chlorobenz[a]anthracene and 7-bromobenz[a]anthracene in the neonatal B6C3F1 male mouse. Cancer Lett. 101, 37-42.

Metabolic Activation of Riddelliine (65) Zhan, D.-J., Chiu, L.-H., Von Tungeln, L. S., Cheng, E., HerrenoSaenz, D., Evans, F. E., Heflich, R. H., and Fu, P. P. (1997) Characterization of DNA adducts in Chinese hamster ovary cells created with mutagenic doses of 1- and 3-nitrosobenzo[a]pyrene and the trans-7,8-diol-anti-9,10-epoxides of 1- and 3-nitrobenzo[a]pyrene. Mutat. Res. 379, 43-52. (66) Fu, P. P., Von Tungeln, L. S., Chiu, L.-H., Zhan, D.-J., Deck, J., and Wang, J.-C. (1998) Structure, tumorigenicity, microsomal metabolism, and DNA binding of 7-nitrodibenz[a,h]anthracene. Chem. Res. Toxicol. 11, 937-945.

Chem. Res. Toxicol., Vol. 14, No. 1, 2001 109 (67) Yi, P., Zhan, D.-J., Samokyszyn, V. M., Doerge, D. R., Evans, F. E., and Fu, P. P. (1997) Synthesis and 32P-postlabeling/HPLC separation of diastereomeric 1,N2-(1,3-propano)-2′-deoxyguanosine 3′-phosphate adducts formed from 4-hydroxy-2-nonenal. Chem. Res. Toxicol. 10, 1259-1265.

TX000150N