Pyrrolizidine Alkaloid-Derived DNA Adducts as a Common Biological

Aug 12, 2013 - Qingsu Xia , Yuewei Zhao , Ge Lin , Frederick A. Beland , Lining Cai , and ... John A. Edgar , Russell J. Molyneux , and Steven M. Cole...
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Pyrrolizidine Alkaloid-Derived DNA Adducts as a Common Biological Biomarker of Pyrrolizidine Alkaloid-Induced Tumorigenicity Qingsu Xia,† Yuewei Zhao,† Linda S. Von Tungeln,† Daniel R. Doerge,† Ge Lin,‡ Lining Cai,§ and Peter P. Fu*,† †

National Center for Toxicological Research, Jefferson, Arkansas 72079, United States School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR § Biotranex LLC, Monmouth Junction, New Jersey 08852, United States ‡

S Supporting Information *

ABSTRACT: Pyrrolizidine alkaloid-containing plants are the most common poisonous plants affecting livestock, wildlife, and humans. The U.S. National Toxicology Program (NTP) classified riddelliine, a tumorigenic pyrrolizidine alkaloid, as “reasonably anticipated to be a human carcinogen” in the NTP 12th Report on Carcinogens in 2011. We previously determined that four DNA adducts were formed in rats dosed with riddelliine. The structures of the four DNA adducts were elucidated as (i) a pair of epimers of 7-hydroxy-9-(deoxyguanosin-N2-yl)dehydrosupinidine adducts (termed as DHP-dG-3 and DHP-dG-4) as the predominant adducts; and (ii) a pair of epimers of 7-hydroxy-9-(deoxyadenosin-N6-yl)dehydrosupinidine adducts (termed as DHP-dA-3 and DHP-dA-4 adducts). In this study, we selected a nontumorigenic pyrrolizidine alkaloid, platyphylliine, a pyrrolizidine alkaloid N-oxide, riddelliine N-oxide, and nine tumorigenic pyrrolizidine alkaloids (riddelliine, retrorsine, monocrotaline, lycopsamine, retronecine, lasiocarpine, heliotrine, clivorine, and senkirkine) for study in animals. Seven of the nine tumorigenic pyrrolizidine alkaloids, with the exception of lycopsamine and retronecine, are liver carcinogens. At 8−10 weeks of age, female F344 rats were orally gavaged for 3 consecutive days with 4.5 and 24 μmol/kg body weight test article in 0.5 mL of 10% DMSO in water. Twenty-four hours after the last dose, the rats were sacrificed, livers were removed, and liver DNA was isolated for DNA adduct analysis. DHP-dG-3, DHP-dG-4, DHP-dA-3, and DHP-dA-4 adducts were formed in the liver of rats treated with the individual seven hepatocarcinogenic pyrrolizidine alkaloids and riddelliine N-oxide. These DNA adducts were not formed in the liver of rats administered retronecine, the nontumorigenic pyrrolizidine alkaloid, platyphylliine, or vehicle control. These results indicate that this set of DNA adducts, DHP-dG-3, DHPdG-4, DHP-dA-3, and DHP-dA-4, is a common biological biomarker of pyrrolizidine alkaloid-induced liver tumor formation. To date, this is the first finding that a set of exogenous DNA adducts are commonly formed from a series of tumorigenic xenobiotics.

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INTRODUCTION

heliotridine-type pyrrolizidine alkaloid N-oxides are also natural plant constituents with quantities nearly equal to their corresponding parent pyrrolizidine alkaloids present in numerous plant species (Figure 1).6 Pyrrolizidine alkaloid Noxides, such as riddelliine N-oxide, monocrotaline N-oxide, and retrorsine N-oxide, have been demonstrated to be reduced enzymatically to the corresponding pyrrolizidine alkaloids in vitro.6,13,14 Platynecine-type pyrrolizidine alkaloids do not contain a double bond at the C1 and C2 positions of the necine base and are either weakly toxic or nontoxic.6,15 Pyrrolizidine alkaloids require metabolic activation to exert their toxicity, including tumorigenicity. It has been shown that liver microsomal metabolism of retronecine-type and heliotridine-type pyrrolizidine alkaloids produce the racemic DHP ((±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine), rather than the optically active 7-R-enantiomer DHR ((−)-R-

Pyrrolizidine alkaloids, produced by plants as secondary metabolites, are common constituents of hundreds of plant species from different botanical families present in many geographical areas around the world.1−8 It is probable that pyrrolizidine alkaloid-containing plants are the most common poisonous plants affecting livestock, wildlife, and humans.1,6−9 Pyrrolizidine alkaloids consist of a necine base and a necic acid. There are four types of necine bases, platynecine, retronecine, heliotridine, and otonecine (Figure 1). The retronecine-, heliotridine-, and otonecine-type pyrrolizidine alkaloids have a double bond at the C1 and C2 positions of the necine base and exhibit high levels of toxicity, including hepatotoxicity and carcinogenicity.2,6 The pyrrolizidine alkaloids that exhibit the most potent genotoxicity and tumorigenicity are the macrocyclic diester pyrrolizidine alkaloids, followed by monoester pyrrolizidine alkaloids.2,6 Retronecinetype pyrrolizidine alkaloids are the most abundant and therefore are the most studied.2,5−8,10−12 Retronecine- and © 2013 American Chemical Society

Received: July 2, 2013 Published: August 12, 2013 1384

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Figure 1. Structures of the necine bases of pyrrolizidine alkaloids, some of the most commonly studied pyrrolizidine alkaloids, and the bases of dehydropyrrolizidine alkaloids.

6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine; dehydroretronecine) or 7-S-enantiomer DHH ((+)-S-6,7-dihydro7-hydroxy-1-hydroxymethyl-5H-pyrrolizine) (Figure 1).16,17 Since the first pyrrolizidine alkaloid, retrorsine, was found to induce liver tumors in experimental animals in 1954,18 a series of tumorigenic pyrrolizidine alkaloids was subsequently identified; all belong to retronecine-, heliotridine-, and otonecine-type pyrrolizidine alkaloids.5,6 It is a concern that humans are exposed to genotoxic and tumorigenic pyrrolizidine alkaloids, and in 1989, the International Programme on Chemical Safety (IPCS) determined that pyrrolizidine alkaloids are a threat to human health and safety.5

The mechanism of tumor formation induced by pyrrolizidine alkaloids was not known for about half a century until in 2001 we determined that riddelliine, a representative pyrrolizidine alkaloid, induces liver tumors (i.e., mainly hemangiosarcoma in male and female rats and male mice) through a genotoxic mechanism mediated by a set of exogenous DHP-derived DNA adducts.19 By 32P-postlabeling/HPLC analysis,20 the levels of the DHP-derived DNA adducts correlated closely with the tumorigenic potencies in rats fed different doses of riddelliine.19,21,22 The levels of DHP-derived DNA adducts formed in vivo in hepatic endothelial cells, the cells of origin for the hemangiosarcomas, were significantly greater than in 1385

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Figure 2. Proposed metabolic activation pathway of riddelliine leading to liver tumor formation.

parenchymal cells, which well correlated with the preferential induction of liver hemangiosarcomas by riddelliine.21 These results were consistent with the finding by Mei et al.23 that in transgenic Big Blue rats, riddelliine induced mutations at guanine bases mainly in liver endothelial cells, not parenchymal cells. Human liver microsomal metabolism of riddelliine generated a metabolic pattern and DNA adduct profile that were very similar to those from rat liver, which indicated that the results of in vivo and in vitro mechanistic studies with experimental animals are highly relevant to humans and that riddelliine can be genotoxic to humans via DHP-derived DNA adduct formation.24 Partly because of these mechanistic findings, the U.S. National Toxicology Program classified riddelliine as “reasonably anticipated to be a human carcinogen” in the NTP 12th Report of Carcinogens in 2011.25 The major limitation associated with the 32P-postlabeling/ HPLC method for DNA adduct analysis is the lack of structural information for the DNA adducts. Subsequently, an HPLC-ESMS/MS methodology was developed for the identification and quantitation of DHP-derived DNA adducts in vivo and in vitro.1 This HPLC-ES-MS/MS method was accurate and precise for quantification of the levels of DHP-2′-deoxyguanosine (DHPdG) and DHP-2′-deoxyadenosine (DHP-dA) adducts by multiple reaction monitoring (MRM) analysis in the presence

of known quantities of isotopically labeled DHP-dG and DHPdA internal standards. When applied to liver samples from rats treated with the pyrrolizidine alkaloids riddelliine and monocrotaline (Figure 1), we determined that four DNA adducts, designated as DHP-dG-3, DHP-dG-4, DHP-dA-3, and DHP-dA-4, were formed. The other four adducts, DHP-dG-1, DHP-dG-2, DHP-dA-1, and DHP-dA-2, which were previously believed to be DNA adducts formed in vivo as analyzed by the 32 P-postlabeling/HPLC method,19 were not present (Figure 2). Therefore, this HPLC-ES-MS/MS method provided significant new information regarding the mechanism of DNA adduct formation.1 However, due to the lack of an adequate amount of authentic standards, the structures of DHP-dA-3 and DHP-dA4 were not determined unequivocally, and the structural assignment for DHP-dG-4 required confirmation.1 Subsequently, an improved synthetic method was developed to prepare these DNA adducts for full structural elucidation by mass spectrometry and NMR spectroscopy.26 The structures of the four DNA adducts were elucidated: (i) DHP-dG-3 and DHP-dG-4, the predominant adducts, were a pair of epimers of 7-hydroxy-9-(deoxyguanosin-N2-yl)-dehydrosupinidine adducts; and (ii) DHP-dA-3 and DHP-dA-4 adducts, the minor adducts, were a pair of epimers of 7-hydroxy-9-(deoxyadenosinN6-yl) dehydrosupinidine adducts (Figure 2).26 We also 1386

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determined that DHP-dG-3 and DHP-dG-4, as well as DHPdA-3 and DHP-dA-4, were interconvertible. This unequivocal DNA adduct structural elucidation enabled us to find that cellular DNA preferentially binds to the reactive pyrrolic metabolite, dehydroriddelliine, at the C9 position of the necine base, rather than at the C7 position. The study of Zhao et al.26 represented the first report with detailed structural assignments of pyrrolizidine alkaloid-derived DNA adducts that are involved in pyrrolizidine alkaloid tumor induction at the molecular level. Consequently, a mechanism of tumor initiation by pyrrolizidine alkaloids was proposed (Figure 2).26 We have hypothesized that this set of DHP-derived DNA adducts is a potential common biomarker of tumorigenicity induced by pyrrolizidine alkaloids and pyrrolizidine alkaloid Noxides.2 Our in vitro preliminary data obtained from liver microsomal metabolism of several pyrrolizidine alkaloids analyzed by 32P-postlabeling/HPLC supported this hypothesis.17,27,28 Since these were in vitro studies, not in vivo, and little information of the DNA adduct structures was provided by 32P-postlabeling analysis, it was difficult to make definitive conclusions on the mechanism at the molecular level. The LC/ MS/MS analysis allowed us to characterize the structures of the DNA adducts and quantify the DNA adducts formed in vivo.1 In the present study, we quantify these four DNA adducts (e.g., DHP-dG-3 and DHP-dG-4, DHP-dA-3, and DHP-dA-4 adducts) that are formed in vivo in the liver of rats administered different types of tumorigenic pyrrolizidine alkaloids. We selected a set of nine tumorigenic pyrrolizidine alkaloids, one pyrrolizidine alkaloid N-oxide (riddelliine N-oxide), and one nontumorigenic pyrrolizidine alkaloid for study. The selected nine tumorigenic pyrrolizidine alkaloids are (i) five retronecinetype pyrrolizidine alkaloids, riddelliine, retrorsine, monocrotaline, lycopsamine, and retronecine, the necine base (Figure 1); (ii) two heliotridine-type pyrrolizidine alkaloids, lasiocarpine and heliotrine; and (iii) two otonecine-type pyrrolizidine alkaloids, clivorine and senkirkine (Figure 1). Among these nine tumorigenic pyrrolizidine alkaloids, six are diesters, two are monoesters, and one necine base without an ester linkage (Figure 1). All nine of these pyrrolizidine alkaloids, with the exception of lycopsamine and retronecine, have been determined to induce liver tumors in rats (Table 1). The nontumorigenic pyrrolizidine alkaloid platyphylliine (a mixture of 70% platyphylliine and 30% neoplatphylliine) was also included for study (Figure 1).

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Table 1. Carcinogenicity of Riddelliine, Monocrotaline, Lycopsamine, Retronecine, Lasiocarpine, Clivorine, Senkirkine, and Heliotrine Determined in Rats pyrrolizidine alkaloid riddelliine

monocrotaline retrorsine

lycopasamine retronecine lasiocarpine

heliotrine

clivorine senkirkine

family (major plant genus)

liver tumor incidence

Retronecine-Type Pyrrolizidine Alkaloids Compositae (Senecio) one year: M 1/4, F 1/12 two years: M 43/50, F 38/50 Leguminosae (Crotalaria) one year: M 10/50 one year: M 5/60 Compositae (senecio) one year: m 4/10, f 1/4 one year: f 2/4 Boraginaceae (amsinckia) nonea Leguminosae (Crotalaria) noneb Heliotridine-Type Pyrrolizidine Alkaloids Boraginaceae one year: M 11/18 (Heliotropium) one year: M 16/20 two years: M 13/24, F 7/9 Boraginaceae two years: M 1/6 (Heliotropium) Otonecine-Type Pyrrolizidine Alkaloids Compositae (Ligularia) one year: M/F 2/12 Compositae (Tussilago, one year: M 9/20 Senecio, Petasites)

refs 29 30 31 32 18 29 33 and 34 35 36 37 38 39

40 41

a Induced islet cell adenoma and bladder papillary tumor. bInduced spinal cord tumor.

Cerilliant Corp. (Round Rock, TX). Clivorine was prepared as previously described.28,43 Platyphylliine (a mixture of 70% platyphylliine and 30% neoplatphylliine) was a gift from Professor H. S. Chen at the Department of Phytochemistry, The Secondary Military Medical University, Shanghai, China. Retronecine was prepared by barium hydroxide catalyzed hydrolysis of monocrotaline. Riddelliine N-oxide was synthesized as previously described.13,44 All chemicals used for the animal in vivo study were analyzed by HPLC and found to be >97% pure. DHP-[15N5]dG-1, DHP-[15N5]dG-2, DHP-[15N5,13C10]dA-1, and DHP-[15N5,13C10]dA-2 adducts were prepared by reaction of dehydroretronecine (DHR) with [15N5]dG and [15N5,13C10]dA, respectively, as previously described.1 All solvents used were of HPLC grade. HPLC Instrumentation for Separation of Chemicals and Metabolites. A Waters HPLC system, consisting of a Model 600 controller, a Model 996 photodiode array detector, and a 600 pump, was used for the separation and purification of pyrrolizidine alkaloids, metabolites of senkirkine, and the DHP-derived DNA adducts. Treatment of Rats with Pyrrolizidine Alkaloids. Procedures involving the care and handling of rats were reviewed and approved by the National Center for Toxicological Research (NCTR) Laboratory Animal Care and Use Committee. Female F344 rats were obtained from the NCTR breeding colony as weanlings and maintained on a 12h light−dark cycle. Eleven chemicals, riddelliine, retrorsine, monocrotaline, lycopsamine, retrornecine, riddelliine N-oxide, lasiocarpine, heliotrine, senkirkine, clivorine, and platyphylliine (a mixture of 70% platyphylliine and 30% neoplatphylliine) were used for this study. At 8−10 weeks of age, 4 female rats per group were orally gavaged for 3 consecutive days with the test article at daily doses of 4.5 and 24 μmol kg body weight in 0.5 mL of 10% DMSO in water, with the exception that retrorsine was administered only at 24 μmol/kg body weight. Rats for the vehicle control group were treated with 10% DMSO in water. Twenty-four hours after the last dose, the rats were sacrificed by exposure to CO2 followed by exsanguination. Livers were removed, rinsed with cold saline, and stored at −70 °C before DNA was isolated

EXPERIMENTAL PROCEDURES

Caution: Riddelliine, retrorsine, monocrotaline, lycopsamine, retronecine, lasiocarpine, clivorine, senkirkine, heliotrine, and riddelliine N-oxide are carcinogenic in laboratory animals. They should be handled with extreme care, using proper personal protective equipment and a wellventilated hood. Chemicals. Monocrotaline, retrorsine, troleandomycin (TAO), 2′deoxyguanosine (dG), 2′-deoxyadenosine (dA), β-nicotinamide adenine dinucleotide 2′-phosphate, reduced (NADPH), micrococcal nuclease, spleen phosphodiesterase, and nuclease P1 were purchased from the Sigma Chemical Co. (St. Louis, MO). [15N5]dG and [15N5,13C10]dA were purchased from Cambridge Isotope Laboratories (Tewksbury, MA). Riddelliine was obtained from Dr. Po-Chan, National Toxicology Program (NTP). Riddelliine-modified rat liver DNA was obtained from female F344 rats treated by gavage with riddelliine in a study conducted by the NTP.42 Lasiocarpine was a gift from Dr. John A. Edgar, CSIRO Livestock Industries, Australia.27 Heliotrine was purchased from Accurate Chemical & Scientific Corporation (Westbury, NY). Senkirkine was obtained from ChromDex (Irvine, CA, USA), and lycopsamine was purchased from 1387

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Figure 3. Mass spectra of (top) DHP-[15N5]dG-3 and (bottom) DHP-[15N5]dG-4. Inserted is the HPLC separation of DHP-[15N5]dG-3 and DHP[15N5]dG-4. Metabolism of Senkirkine by Rat Liver Microsomes in the Presence of P450 3A Enzyme Inhibitor. The metabolism of senkirkine by male F344 rat liver microsomes in the presence of an enzyme inhibitor, TAO, was similarly conducted as described above. A 1 mL incubation mixture containing 100 mM sodium phosphate buffer (pH 7.6), 5 mM magnesium chloride, 1 mM NADPH, and 1 mg of microsomal protein was preincubated with 40 nmol of TAO (in DMSO) at 37 °C for 10 min. Senkirkine was then added, and the resulting incubation mixture was incubated at 37 °C for an additional 30 min. Control incubations in the presence of DMSO were also conducted in parallel. HPLC-ES-MS/MS Analysis of DHP-Derived DNA Adducts Formed In Vivo: Quantification of DHP-DNA Standards. Purified samples of the DHP-dG-1, DHP-dG-2, DHP-dG-3, and DHP-dG-4 adduct standards were quantified spectrophotometrically based on the UV molar extinction coefficients 1.70 × 104 and 1.72 × 104 M−1cm−1 at 256 nm (pH 7.0) as reported by Robertson.46 DHP-dA-1, DHP-dA2, DHP-dA-3, and DHP-dA-4 adduct standards were also quantified spectrophotometrically using the following experimentally determined UV molar extinction coefficients: 2.47 ± 0.26 × 104 and 2.48 ± 0.14 × 104 M−1cm−1, at 271 nm in water and 2.21 ± 0.23 × 104 and 2.18 ± 0.12 × 104 M−1cm−1 in methanol at 270 nm. Isotopically labeled standards DHP-dGs and DHP-dAs were used as the internal standards to quantify the responding adducts. The concentrations of the isotopically labeled adduct standards for DHP-dG-3, DHP-dG-4, DHP-dA-3, and DHP-dA-4 were quantified by HPLC-UV (256 nm) by comparison to the unlabeled standards. After adding 50 fmol of each internal standard, the samples were injected on a LC-MS/MS system for quantitative analyses. Quantitation of DHP-dG and DHP-dA Adducts by LC/MS/MS Analysis Liquid Chromatography. A Shimadzu Prominence HPLC

for DNA adduct analysis. DNA samples were extracted using a Blood & Cell Culture DNA Isolation Kit (QIAGEN Inc., Valencia, CA) according to the manufacturer’s instructions. The concentration of the DNA was determined spectrophotometrically. DNA samples (typically ∼100 μg) obtained from the liver of rats treated with chemicals or vehicle were enzymatically hydrolyzed to nucleosides with micrococcal nuclease, spleen phosphodiesterase, and nuclease P1 as previously described.45 Synthesis of Isotopically Labeled DHP-dG-3, DHP-dG-4, DHP-dA-3, and DHP-dA-4 Adducts. Following the previously described procedure for the synthesis of DHP-dG-3, DHP-dG-4, DHP-dA-3, and DHP-dA-4 adducts,1 DHP-[15N5]dG-3, DHP[15N5]dG-4, DHP-[15N5,13C10]dA-3, and DHP-[15N5,13C10]dA-4 adducts were prepared by the reaction of dehydromonocrotaline (6.0 mg, 0.02 mmol) with [ 15 N 5 ]dG (5 mg, 0.02 mmol) and [15N5,13C10]dA (5 mg, 0.02 mmol). Metabolism of Senkirkine by Rat Liver Microsomes. The metabolism of senkirkine by female 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 NADPH, 0.5 mM senkirkine, and 2 mg of rat liver microsomal protein at 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 metabolites present in the incubation mixture were analyzed by reverse phase HPLC employing a Prodigy 5 μm ODS column (4.6 × 250 mm, Phenomenex, Torrance, CA). HPLC conditions: monitored at 220 nm, with a flow rate of 1 mL/ min; gradient program, 0−10 min, 20 mM ammonium acetate buffer (pH 7); 10−40 min, from 20 mM ammonium acetate buffer (pH 7.0) to 50% methanol in the buffer. 1388

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Table 2. Levels of DHP-dG and DHP-dA Adducts Formed in the Livers of Female Rats Dosed with Pyrrolizidine Alkaloids for Three Consecutive Daysa levels of DHP-dG and DHP-dA/108 nucleotides PA control retrorsine lasiocarpine riddelline monocrotarine riddelliine N -oxide senkikine heliotrine clivorine lycopsamine retronecine platyphylliine

b

dose (μmol/kg b.w./day)

DHP-dG-3

DHP-dG-4

DHP-dA-3

DHP-dA-4

total DHP-dG and DHP-dA

DMSO 24 4.5 24 4.5 24 4.5 24 4.5 24 4.5 24 4.5 24 4.5 24 4.5 24 4.5 24 4.5 24 4.5