Species-Specific Differences in the in Vitro Metabolism of

Sep 22, 2015 - Rabbits, sheep, goats, and guinea pigs are resistant, whereas humans, pigs, rats, mice, cattle, and horses are species that are suscept...
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Species-Specific Differences in the in Vitro Metabolism of Lasiocarpine Muluneh M. Fashe,† Risto O. Juvonen,† Aleksanteri Petsalo,† Juha Ras̈ an̈ en,‡,§ and Markku Pasanen*,† †

School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio, Finland Department of Obstetrics and Gynecology, University of Oulu, FI-90220 Oulu, Finland § Department of Obstetrics and Gynecology, Kuopio University Hospital and University of Eastern Finland, FI-70029 Kuopio, Finland ‡

S Supporting Information *

ABSTRACT: There are species-related differences in the toxicity of pyrrolizidine alkaloids (PAs) partly attributable to the hepatic metabolism of these alkaloids. In this study, the metabolism of lasiocarpine, a potent hepatotoxic and carcinogenic food contaminant, was examined in vitro with human, pig, rat, mouse, rabbit, and sheep liver microsomes. A total of 12 metabolites (M1−M12) were detected with the human liver microsomes, of which M1, M2, M4, and M6 were unstable in the presence of reduced glutathione (GSH). With the exception of M3 and M8, the formation of all metabolites of lasiocarpine was catalyzed by CYP3A4 in humans. Tandem mass spectra (MS/MS) detected several new metabolites, termed M4−M7; their toxicological significance is unknown. M9 (m/z 398), identified as a demethylation product, was the main metabolite in all species, although the relative dominance of this metabolite was lower in humans. The level of the reactive metabolites, as measured by M1 ((3H-pyrrolizin-7-yl)methanol) and the GSH conjugate, was higher with the liver microsomes of susceptible species (human, pig, rat, and mouse) than with the species (rabbit and sheep) resistant to PA intoxication. In general, in addition to the new metabolites (M4−M7) that could make humans more susceptible to lasiocarpine-induced toxicity, the overall metabolite fingerprint detected with the human liver microsomes differed from that of all other species, yielding high levels of GSH-reactive metabolites.

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INTRODUCTION Lasiocarpine is one of the most toxic pyrrolizidine alkaloids (PAs); these compounds are important secondary metabolites present in many flowering plants.1 Lasiocarpine is widely produced by the members of the Boraginaceae family,2 such as Heliotropium (e.g., H. eichwaldii Steud. ex. Dc., H. europaeum, H. indicum. L., and H. lasiocarpum Fisch. et. Mey) and Symphytum (e.g., S. off icinale Linn.) species. These plants are commonly used as herbal medicines, and they can also contaminate human foods such as wheat.3−7 Exposure to lasiocarpine, as well as other PAs, has been known to cause fatal liver veno-occlusive disease in humans and animals.8−10 For instance, lasiocarpine was detected in the wheat flour that caused a human poisoning outbreak in Afghanistan (2007−2008).6 Furthermore, the alkaloid is a carcinogen (Group 2B) capable of inducing hepatocellular tumors and angiosarcomas of the liver as well as malignant tumors of the skin in rats.11−13 On the basis of its LD50 value, lasiocarpine (77 mg/kg) is the second most potent acutely lethal toxin of the known PAs after retrorsine (34 mg/ kg) in rats given a single intraperitoneal dose.4 Significant variations have been observed between species with respect to the toxicities of PAs. Rabbits, sheep, goats, and guinea pigs are resistant, whereas humans, pigs, rats, mice, cattle, and horses are species that are susceptible to PA intoxication.14−16 Several factors are known to contribute to the © XXXX American Chemical Society

species-related differences toward PA toxicity, although the balance between metabolic bioactivation and deactivation of PAs is often considered to be the major source of the variability.14 In fact, metabolic bioactivation is a primary requirement for the development of PA toxicity.4,17,18 For instance, rats are susceptible to PA intoxication because of the high rate of pyrrolic metabolite formation and their inadequate carboxylesterase-mediated hydrolysis,19 whereas guinea pigs are resistant since they possess a high capacity for hydrolytic detoxification of hepatotoxic PAs.20 Like all other hepatotoxic PAs, lasiocarpine is toxic only after its metabolic conversion to the toxic intermediate, known as dehydrolasiocarpine.5,21 Dehydrolasiocarpine and other putative didehydropyrrolizidine alkaloids (the pyrrolic esters) are very reactive; they attack nucleophilic macromolecules such as DNA and proteins, eliciting severe toxicities, including liver veno-occlusive disease and tumors.14 Because of their extreme instability, the pyrrolic esters have not yet been identified either in vivo or in vitro;22 instead, they may bind to nucleophilic macromolecules such as DNA, conjugate with glutathione (GSH), or hydrolyze to a more stable but less reactive racemic mixture of dehydroretronecine and dehydroheliotridine, a DHP Received: June 16, 2015

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(3), sheep liver (8), or sheep fetus liver (8) in 100 mM potassium phosphate buffer, pH 7.4, at 37 °C for 10 min. The reaction was initiated by addition of 40 μL of the NADPH regenerating system. Immediately after the start of the reaction, 30 μL of the sample was withdrawn and added to 90 μL of acetonitrile. This was designated as the 0 min incubation sample, and similar procedures were repeated at 15, 30, 60, and 120 min after the start of the reaction. The blanks were devoid of the NADPH regenerating system. The reactivity of lasiocarpine metabolites (i.e., GSH conjugation) was studied by including 4.4 mM reduced GSH to the reaction mixture for 120 min at 37 °C, whereas the stability of each metabolite was determined under the same reaction conditions as those for human liver microsomes for 30 min. The role of human cytochrome P450 3A4 (CYP3A4) in the metabolism of lasiocarpine was studied by replacing liver microsomes with the CYP3A4 enzyme (2 pmol) under the reaction conditions described above for 30 min. The inhibition of CYP3A4- or human liver microsome-catalyzed metabolism of lasiocarpine by ketoconazole was studied by including 0.0, 0.1, 1.0, or 10 μM of the inhibitor into the reaction mixture. At the end of each incubation period, the reaction was terminated by acetonitrile and the samples were immediately centrifuged (Mini Spin, Eppendorf, Hamburg, Germany) at 10 000g for 10 min; then, the supernatants were collected and directly analyzed by a liquid chromatography/mass spectrometry (LC-MS) system. HPLC Conditions. A gradient elution with two HPLC solvents (A, ammonium acetate; B, acetonitrile) was used, i.e., 5% solvent B at 0 min was linearly increased to 100% in 10 min and reduced back to 5% in 0.1 min. Between each consecutive injection, the system was allowed to equilibrate for 3 min. The solvent flow rate and sample injection volume were 0.4 mL/min and 2 μL, respectively. Identification of Lasiocarpine Metabolites. The identification of all of the lasiocarpine metabolites was performed with an Agilent 6540 Q-TOF LC-MS system (Agilent Technologies, Palo Alto, CA, USA). The Q-ToF instrument was coupled with LC (Agilent 1290 series rapid resolution, Waldbronn, Germany), and the chromatographic separation was achieved with an Agilent Zorbax SB-C18 column (2.1 × 50 mm, 1.8 μm particle size) supplemented with an Agilent Infinity in-line filter (0.2 μm). Other LC conditions were as described in the HPLC Conditions section above. Accurate masses for all metabolites and their MS/MS fragments were acquired, and the mass measurement errors between the predicted mass and the measured accurate masses were calculated for each metabolite. The QToF MS conditions were as follows: positive ESI; capillary voltage, 3.5 kV; fragmentor voltage, 100 V; sheath gas flow rate, 11 L/min at 350 °C (nitrogen); drying gas flow rate, 10 L/min at 325 °C (nitrogen); and nebulizer, adjusted to a 45 psi. As a lock mass, Agilent reference mass solution (m/z 121.05087 and 922.00979) was used. Semiquantitative LC-MS Analysis of Lasiocarpine Metabolites. Sample introduction into the ESI-MS was achieved by using an Agilent 1200 rapid resolution HPLC system with the chromatographic separation performed with a reverse-phase column (Agilent ZORBAX Eclipse XDB-C18 4.6 × 50 mm, 1.8 μM particle size). The other LC conditions were as described in the HPLC Conditions section above. The HPLC column oven temperature was 50 °C. Semiquantitative analysis of the metabolites was performed on a 6410 series triple quadrupole MS (Agilent Technologies, Palo Alto, CA, USA) equipped with an ESI interface in positive mode. Multiple reaction monitoring (MRM) was used for the acquisition of peak area as follows: M1 and M2 (m/z: 136 → 80); M3 (m/z: 238 → 120); M4 and M5 (m/z: 312 → 152); M6 and M7 (m/z: 326 → 152); M8 (m/z: 330 → 138); M9 (m/z: 398 → 120); lasiocarpine (m/z: 412 → 120); M10 (m/z: 428 → 254); M11 (m/z: 428 → 120); and M12 (m/z: 444 → 152). The MS conditions were as follows: sheath gas temperature, 300 °C; gas flow, 10 L/min; nebulizer, 40 psi; capillary voltage, 4.0 kV; and fragmentor, 100 V. The LC-MS peak area and MS/MS data were used in the analysis and interpretation of the results. LC-MS Analysis of the GSH Conjugate. The GSH conjugate of the reactive lasiocarpine metabolites was analyzed by LTQ MS (ThermoFinnigan linear ion trap, Thermo Fisher Scientific Inc., Waltham, MA, USA). The MS instrument was coupled to an Agilent 1200 HPLC system (Agilent Technologies, Waldbronn, Germany),

([(±)6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine]).14 Moreover, recently we were able to produce dehydroretrorsine by oxidizing retrorsine in an electrochemical cell; this intermediate was rapidly degraded to a more stable metabolite known as (3H-pyrrolizin-7-yl)methanol.23 (3HPyrrolizin-7-yl)methanol, a potentially toxic metabolite of retrorsine, reacted readily with GSH to form the previously identified GSH conjugates: mono- and di-GSH-DHP.22 In addition to this small reactive metabolite, we also utilized the electrochemical oxidation method to reveal that retrorsine could be oxidized to several other metabolites.23 Thus, although the oxidation of PAs has been extensively studied,4 there are still unanswered questions about the metabolic bioactivation of these compounds, such as for toxic lasiocarpine. In addition to the reactive intermediate dehydrolasiocarpine, earlier studies have also demonstrated that lasiocarpine can be metabolized to lasiocarpine N-oxide by rat liver microsomes.5,21 However, the metabolism of lasiocarpine in humans as well as many other species has not been studied in a comprehensive manner. Therefore, the three main objectives of this study were (1) to study if lasiocarpine could be metabolized to (3Hpyrrolizin-7-yl)methanol and to other potentially reactive metabolites, (2) to elucidate the major in vitro route of metabolic elimination of lasiocarpine, and (3) to compare the formation of these in vitro metabolites of lasiocarpine in human, pig, rat, mouse, rabbit, and sheep liver microsomes.

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MATERIALS AND METHODS

Caution: Lasiocarpine (CASRN: 303-34-4) and echimidine (CASRN: 520-68-3) are hazardous. These alkaloids are known hepatotoxins and should be handled with due care. Chemicals and Reagents. Lasiocarpine and echimidine (>95% pure) were purchased from PhytoLab (Vestenbergsgreuth, Germany); glutathione, ketoconazole, ammonium acetate, and potassium phosphate were obtained from Sigma-Aldrich (Steinheim, Germany); nicotinamide adenine dinucleotide phosphate (NADP+) was purchased from Roche Diagnostics (Mannheim, Germany); and acetonitrile and methanol were obtained from VWR International (Leuven, Belgium). Pure water (Milli-Q) was purified by Merck Millipore (Billerica, MA). Ten millimolar lasiocarpine was prepared in methanol and stored below −20 °C on a daily basis. The NADPH regenerating system was composed of 1.12 mM NADP+, 12.5 mM MgCl2, 12.5 mM MnCl2, 16.8 mM isocitric acid, 0.056 mM KCl, and 15 U of isocitric acid dehydrogenase in 188 mM Tris-HCl buffer, pH 7.4. Liver Microsomes. The collection of liver samples from human (HL22, female), pigs (female), rats (female, Sprague−Dawley), mice (female, DBA/2N), and rabbits (male, New Zealand white) has been described in our earlier studies.24,25 Adult sheep (female) and sheep fetus (unknown sex) liver samples were obtained from Oulu University (Oulu, Finland), and the collection of the liver specimen was approved by the Ethics Committee of the University (no. ESAVI/ 3510/04.10.03/2011). Pooled human liver microsomes and cDNAexpressed human wild-type CYP3A4 Supersomes (with b5) were purchased from BD Biosciences Discovery Labware (Bedford, MA). The HL22 liver sample donor had a history of drug use such as dexamethasone, nizatidine, and phenytoin 24 h before death from intracerebral hemorrhage. All other liver samples were obtained from untreated animals. Liver microsomes were prepared as described previously,26 and the protein concentration was determined by the Bradford method.27 With the exception of HL22, pooled samples of liver microsomes were used during the in vitro incubation studies. In Vitro Metabolic Assay. Ten micromolar lasiocarpine was preincubated (Heildoph Incubator 1000, Germany) in a 200 μL reaction mixture containing water and ≈3.7 μg of microsome protein from pooled human liver, individual human liver (HL22), pig liver (pool of three 3 pig livers), rat liver (6), mouse liver (10), rabbit liver B

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Chemical Research in Toxicology Table 1. Accurate Mass Data of the in Vitro Metabolites of Lasiocarpine with Human Liver Microsomesa compound

molecular weight

LAS* M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12

411 135 135 237 311 311 325 325 329 397 427 427 443

MS/MS fragments 412, 136, 136, 238, 312, 312, 250, 250, 330, 398, 428, 410, 444,

336, 118, 118, 138, 250, 250, 152, 152, 254, 336, 352, 254, 270,

220, 108, 108, 120, 152, 152, 134, 134, 138, 220, 254, 236, 152,

138, 120 94, 80 94, 80 94 134 134 59 59 120 138, 120 236, 120 138, 120 134, 106

predicted formula

observed mass

predicted mass

error (ppm)

C21H33NO7 C8H9NO C8H9NO C13H19NO3 C15H21NO6 C15H21NO6 C16H23NO6 C16H23NO6 C16H27NO6 C20H31NO7 C21H33NO8 C21H33NO8 C21H33NO9

412.2330 136.0757 136.0757 238.1439 312.1436 312.1441 326.1599 326.1601 330.1911 398.2173 428.2279 428.2279 444.2229

412.2330 136.0757 136.0757 238.1438 312.1442 312.1442 326.1598 326.1598 330.1911 398.2173 428.2279 428.2279 444.2228

0.00 0.00 0.00 −0.42 1.92 0.32 −0.31 −0.92 0.00 0.00 0.00 0.00 −0.23

The accurate mass data were acquired on an ESI-Q-ToF-MS instrument. The mass measurement error was estimated as ((observed mass − predicted mass)/observed mass) × 106. LAS, lasiocarpine a

Figure 1. LC-MS/MS chromatogram of lasiocarpine and its metabolites. Ten micromolar lasiocarpine was incubated with human liver microsomes (∼3.7 μg of protein) in 100 mM potassium phosphate buffer, pH 7.4, in the presence of NADPH for 60 min. M2 was observed only at the higher concentrations of lasiocarpine (≥25 μM); hence, its LC-MS chromatogram was not visible from the 10 μM lasiocarpine incubation (see Figure S2). Data Analysis. Acquisition and analysis of the QToF- and triple quadrupole-MS data were achieved by Agilent MassHunter Workstation software (MassHunter B.04.00). The LTQ data for the GSH conjugate were acquired and analyzed with Xcalibur v.1.4 software.

and the chromatographic separation was achieved on a Hypersil graphite column (100 × 4.6 mm, Cheshire, England). The other LC conditions were as described in the HPLC Conditions section above. The LTQ was set to operate with the MS and MS/MS analyses with a mass scan range of m/z 200−1000 in negative ESI mode. The tuning conditions were as follows: sheath gas flow rate, 30 AU; spray voltage, 3.80 kV; capillary temperature, 250 °C; and capillary voltage, −20 V.

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RESULTS The metabolism of 10 μM lasiocarpine was examined in vitro with the liver microsomes from humans, pigs, rats, mice, C

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Chemical Research in Toxicology Scheme 1. Proposed Molecular Structures and Metabolic Pathways of Metabolites of Lasiocarpine

Identification of the Metabolites of Lasiocarpine. The proposed molecular formula, predicted and measured accurate masses, mass measurement errors, and ESI-MS/MS fragment ions of lasiocarpine metabolites are illustrated in Table 1; known and proposed molecular structures of all metabolites of lasiocarpine and their possible pathways of formation are depicted in Scheme 1. On the basis of the MS and MS/MS data, the 12 metabolites of lasiocarpine were categorized into five major metabolic pathways: dehydrogenation (M1, M2, M4−M7), ester bond cleavage (M3 and M8), demethylation (M9), N-oxidation (M10), and hydroxylation (M11 and M12). Dehydrogenation. M1 had a protonated molecular ion at m/z 136 and produced MS/MS fragment ions at m/z 118, 108, 94, and 80 (Table 1 and Figure S1). It was identified as (3Hpyrrolizin-7-yl)methanol.23 M2 produced identical MS/MS fragment ions as those of M1 (Table 1); hence, it was identified as an isomer of M1, (3H-pyrrolizin-7-yl)methanol. However, M2 was a minor metabolite, and it was observed only at ≥25 μM substrate (lasiocarpine) concentrations (Figure S2).

rabbits, and sheep. The results indicated that lasiocarpine was extensively metabolized by human liver microsomes, and a total of 12 metabolites (M1−M12) could be detected at the end of the 30 min incubation period (Table 1 and Figure 1). Several of the metabolites were also detected after oxidation of lasiocarpine with liver microsomes from all of the species included in this study. In contrast, M4−M6 were either not detected at all or their relative abundance was less than 1% in pig, rat, mouse, rabbit, and sheep liver microsomes. The fastest rate of lasiocarpine oxidation was observed with human HL22 microsomes, followed by mouse liver microsomes, and the slowest rate was observed with rabbit liver microsomes. The order of the in vitro rate of metabolic elimination of lasiocarpine (according to t1/2) was as follows: HL22 (16 min) > mouse (43 min) > pig (87 min) > rat (116 min) > sheep (114 min) > human (pooled) (139 min) > rabbit (347 min). We also studied the metabolism of lasiocarpine with liver microsomes derived from sheep fetus, but the extent of metabolism was insignificant and hence is not reported here. D

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Figure 2. MS/MS spectra of M4−M7. These metabolites showed comparable MS/MS fragment ions at m/z 134 and 152. The MS/MS data were acquired with Q-ToF MS. Although metabolites of pyrrolizidine alkaloids with these types of ESI-MS/MS fragment ions have not been reported from in vitro or in vivo metabolic matrices, electrochemical oxidation of retrorsine yielded products with similar ESI-MS/MS fragment ions23 (see Figure S7).

Ester Bond Cleavage. M3 (m/z 238) produced ESI-MS/MS fragment ions at m/z 138, 120, and 94. Similarly, M8 (m/z 330) displayed ESI-MS/MS fragment ions at m/z 254, 138, and 120 (Table 1 and Figure S1). On the basis of the MS/MS fragment ions and the measured accurate masses, we propose that M3 and M8 were formed after ester bond cleavage at C9 and C7 of lasiocarpine, respectively (Scheme 1). It is also possible that M3 was formed after cleavage of the ester bond of M9 at the C9 position. For instance, echimidine, a stereoisomer of the proposed structure of M9, produced a metabolite at m/z

M4 and M5 (m/z 312) as well as M6 and M7 (m/z 326) exhibited common major MS/MS fragment ions at m/z 134 and 152, indicating that they had similar structural features (Scheme 1). The measured accurate masses indicated that the fragment ions at m/z 134 and 152 originated from the necine base moiety of lasiocarpine (Table 1 and Figure 2). Since the formation of the dehydrogenation metabolites required both enzyme-catalyzed hydroxylation and spontaneous dehydration, these metabolites were considered to be secondary products of unstable intermediate metabolites (Scheme 1). E

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Figure 3. NADPH- and cytochrome P450 (CYP) enzyme-dependent formation of M3 and M8. (A) NADPH dependence was evaluated by incubating 10 μM lasiocarpine with pooled human liver microsomes (∼3.7 μg of protein) in the presence (reaction) or absence of NADPH (blank). (B) Ketoconazole-induced inhibition of M3 and M8 formation was evaluated by incubating 10 μM lasiocarpine with human liver microsomes in the presence of different concentrations of ketoconazole.

Figure 4. Pattern of in vitro metabolites of lasiocarpine in different species: humans, pigs, rats, mice, rabbits, and sheep. The data (LC-MS peak area) were obtained after incubation of 10 μM lasiocarpine with liver microsomes (∼3.7 μg of protein) from human, pig, rat, mouse, rabbit, and sheep in the presence of NADPH for 30 min at 37 °C. Duplicates of two independent experiments were analyzed.

238, which was identical with M3 both in its LC-MS and ESIMS/MS spectra (Figure S3). Although we observed that the production of both M3 and M8 was NADPH-dependent

(Figure 3a) and that their formation was inhibited by ketoconazole (Figure 3b), neither M3 nor M8 could be detected when the alkaloid was incubated with recombinant F

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Chemical Research in Toxicology human CYP3A4. At 10 μM lasiocarpine, we did not observe the simultaneous cleavage of the C7 and C9 ester bonds, which could have subsequently yielded heliotridine, the necine base of lasiocarpine. Demethylation. The molecular weight of M9 (m/z 398) was 14 Da units lower than that of lasiocarpine, and its major MS/ MS fragment ions were at m/z 336, 220, 138, and 120 (Table 1). Furthermore, the ESI-MS/MS fragment ions of the necine base moieties of M9 and lasiocarpine were identical (Table 1), indicating that a metabolic change had occurred on the necic acid moiety of the alkaloid. Furthermore, in order to determine the molecular structure of the demethylation metabolite, the LC-MS (Figure S4) and ESI-MS/MS (Figure S5) spectra of echimidine and M9 were compared. Thus, on the basis of the accurate mass measurement error and ESI-MS/MS data as well as the structure of the known standard (echimidine), M9 was unequivocally identified as a demethylation product of lasiocarpine, and it has a molecular structure as depicted in Scheme 1. According to the LC-MS peak area, M9 was the predominant metabolite in all of the species studied (Figure 4). N-Oxidation. M10 (m/z 428) was 16 Da mass units higher than the parent alkaloid, which is evidence for the addition of an oxygen atom. M10 displayed the characteristic ESI-MS/MS fragment ions (Table 1 and Figure S6) of N-oxide of hepatotoxic PAs;28 hence, it was identified as lasiocarpine Noxide (Scheme 1). Hydroxylation. M11 (m/z 428) was 16 Da mass units higher than lasiocarpine, evidence that there had been addition of an oxygen atom. Furthermore, the metabolite showed the characteristic ESI-MS/MS fragment ions of the PAs, i.e., at m/z 120 and 138 (Table 1 and Figure S1), indicating that the site of oxidation was on the necic acid moiety of the alkaloid. Thus, M11 was tentatively identified as a hydroxyl metabolite of lasiocarpine. M12 (m/z 444) was 32 Da higher than lasiocarpine (m/z 412), pointing to the addition of two oxygen atoms. The absence of MS/MS fragment ions at m/z 120 and 138 (Figure S1) proved that the two oxygen atoms might be added to the necine base moiety of the alkaloid. Effect of GSH on the Stability of Lasiocarpine Metabolites. The stability or reactivity of the metabolites of lasiocarpine was studied by including 4.4 mM reduced GSH in the incubation mixture. The results revealed that the effect of GSH on the formation of M3, M5, M7−M9, M11, and M12 was insignificant. In contrast, the presence of GSH reduced the level of M1, M4, and M6 by over 85% (Figure 5). The level of N-oxide (M10) was reduced by 35%. Previously, we demonstrated that M1 and M2 are reactive toward reduced GSH and produce the typical GSH conjugates of the hepatotoxic PAs,23 whereas M4 and M6 were new metabolites. Thus, M3 and M8−M12 were considered to be nonreactive, whereas M1, M4, and M6 were unstable metabolites of lasiocarpine in the presence of reduced GSH. However, because we did not use other trapping agents, particularly, hard nucleophiles, there could be limitations in the classification of lasiocarpine metabolites as reactive and nonreactive. In the presence of human liver cytosolic fraction in the incubation mixture, both M6 and M7 were unstable (data not shown). Role of CYP3A Enzymes. In order to determine the role of CYP3A4 in the metabolism of lasiocarpine, we examined the metabolism of 10 μM lasiocarpine with recombinant human CYP3A4, and the results confirmed that all metabolites of lasiocarpine except for M3 and M8 could be produced. Furthermore, the presence of 0.1 μM ketoconazole inhibited

Figure 5. Effect of reduced glutathione on the formation of lasiocarpine metabolites. The formation of lasiocarpine metabolites was studied by incubating 10 μM lasiocarpine with human liver microsomes (∼3.7 μg of protein) and NADPH at 37 °C for 30 min in the presence of 4.4 mM reduced GSH. Values are the mean ± standard deviation. Duplicates of two independent experiments were analyzed.

the recombinant human CYP3A4-mediated formation of all metabolites of lasiocarpine by more than 60%. Similarly, the inclusion of 0.1 μM ketoconazole in the incubation mixture of 10 μM lasiocarpine with human liver microsomes caused a greater than 50% reduction in the enzymatic activity toward the formation of all lasiocarpine metabolites. Overall, the results provide convincing evidence that the metabolism of lasiocarpine is mainly catalyzed by the CYP3A enzyme family, in particular, CYP3A4. Metabolism of Lasiocarpine in Different Species. Earlier studies demonstrated that humans, pigs, rats, and mice are susceptible to PA-mediated hepatotoxicity, whereas sheep and rabbits are resistant.14,15 In this study, we investigated the NADPH-dependent oxidation of lasiocarpine in vitro by utilizing the liver microsomes of humans, pigs, rats, mice, rabbits, and sheep. The results revealed that M9 was the main in vitro oxidation metabolite of lasiocarpine in all species and that the highest rate of M9 formation was observed with mouse microsomes, followed by sheep microsomes (Figures 4 and 6). In humans, in addition to M9, other metabolites, such as M1, M7, and M12, were also significant according to their LC-MS peak area (Figure 7). Moreover, the production of M9 was not affected by the presence of reduced GSH. Another significant species-dependent difference found in the metabolism of lasiocarpine was the rate of the formation of M1 ((3H-pyrrolizin-7-yl)methanol), which was highest with HL22 microsomes, followed by mouse (Figure 7a). Similarly, the highest rate of GSH conjugate formation was observed with liver microsomes from PA-susceptible species: humans, pigs, rats, and mice (Figure 7b). Since both M1 and mono-GSHDHP may indirectly measure the rate of dehydrolasiocarpine formation, susceptible species, particularly humans, seemed to produce the toxic metabolite at a higher rate than the resistant species, rabbit and sheep.

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DISCUSSION Earlier studies have revealed that lasiocarpine can be oxidized to dehydrolasiocarpine, a putative toxic metabolite, and nontoxic lasiocarpine N-oxide. 5,28 This study presents a more G

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have shown that lasiocarpine can exist in several binding orientations in the active site of human CYP3A4 because of its flexible structural features, such as open chain necic acids.29 The dehydrogenation route produces the ultimate toxic pyrrolic esters, whereas the N-oxidation and hydrolysis pathways are considered to be the major routes of PA detoxification.14 However, in our study, lasiocarpine N-oxide and the ester bond cleavage metabolites were found to be minor pathways; instead, M9 (the demethylation product) was the major metabolite of lasiocarpine. The formation of M9 was not affected by the presence of reduced GSH in the reaction mixture, indicating that M9 was nonreactive and perhaps the major detoxification metabolite of lasiocarpine. However, since this metabolite possesses all of the essential structural features of hepatotoxic PAs,17 further metabolic bioactivation of M9 to pyrrolic metabolites may not be ruled out in rapid metabolizers such as HL22. For instance, we propose that M9 might be the precursor of M3−M5 (Scheme 1). The dehydrogenation pathway produced six metabolites (M1, M2, M4−M7), of which M1 and M2 produced typical ESI-MS/MS fragment ions (m/z 80, 108, 118, and 136) of the pyrrolic metabolites of PAs.23,30 On the other hand, the new metabolites of lasiocarpine, i.e., M4−M7, exhibited atypical ESI-MS/MS fragment ions at m/z 134 and 152. Since the necine base moieties of hepatotoxic PAs such as lasiocarpine characteristically produce ESI-MS/MS fragment ions at m/z 156, 138, and 120, it is evident that the metabolic biotransformation process had centered on the necine bases of M4−M7. Hence, these metabolites were classified as dehydrogenation pathway metabolites. To the best of our knowledge, metabolites of PAs with these types of ESI-MS/MS fragmentation patterns have not been reported in the literature, although we observed metabolites with similar MS/MS spectra (Figure S7) after electrochemical oxidation of retrorsine.23 While metabolites M1 and M2 are common to all hepatotoxic PAs, the new dehydrogenation pathways metabolites (M4− M7) might be lasiocarpine-specific. Earlier studies showed that the dehydrogenation pathway metabolites, particularly the putative didehydropyrrolizidine (the pyrrolic esters), are responsible for the hepatotoxic and genotoxic carcinogenic actions of PAs such as lasiocarpine.31,32 Although the molecular structures of M4−M7 and their mechanism of formation were not elucidated, the four metabolites displayed some distinctive chemical and spectral features. For instance, M4 and M5 had the same molecular

Figure 6. Formation of the main metabolite, M9, from lasiocarpine with the liver microsomes from different species. Lasiocarpine (10 μM) was incubated with liver microsomes (∼3.7 μg of protein) from humans (HLp and HL), pigs (PL), rats (RL), mice (ML), rabbits (BL), and sheep (SL) in 100 mM potassium phosphate buffer (pH 7.4) for 30 min. The samples were analyzed by LC-MS, and the peak area of M9 from each sample was calculated. Values are the mean ± standard deviation. Duplicates of two independent experiments were analyzed.

comprehensive picture of the oxidative metabolism of lasiocarpine with liver microsomes from humans, pigs, rats, mice, rabbits, and sheep. In summary, a total of 12 metabolites (M1−M12) of lasiocarpine were detected with human liver microsomes, and the demethylation product, M9, was the predominant metabolite in all species. When reduced GSH was present in the incubation medium, the levels of M1, M4, and M6 were substantially reduced, whereas the levels of the others were only mildly influenced. Our earlier study demonstrated that M1 and its minor isomer M2 are common reactive metabolites of retrorsine,23 whereas M4 and M6 seemed to be new reactive metabolites of lasiocarpine. Although identical metabolites of lasiocarpine were observed with the liver microsomes from all of the other species studied, the metabolic fingerprint of human liver microsomes differed from that of the other species in two major respects: (1) the relative amount of the main metabolite, M9, was lower with HL microsomes than with the liver microsomes of other species and (2) more GSHreactive metabolites were formed with HL microsomes. Our results reveal that lasiocarpine is mainly metabolized in vitro through five metabolic pathways, dehydrogenation, ester bond cleavage, demethylation, N-oxidation, and hydroxylation, indicating that several sites on its molecular structure are subjected to oxidation. In fact, by utilizing in silico methods, we

Figure 7. Formation of metabolites M1 (A) and mono-GSH-DHP (B) in different species. Ten micromolar lasiocarpine was incubated with liver microsomes (∼3.7 μg of protein) from humans (HL22 and HLp), mice (ML), pigs (PL), rats (RL), rabbits (BL), and sheep (SL) in 100 mM potassium phosphate buffer (A) or in the presence of 4.4 mM GSH (B). Values are the mean ± standard deviation. Duplicates of two independent experiments were analyzed. H

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metabolite formation.15 The same study also claimed that the higher rate of GSH conjugate formation could be associated with the resistance of these animals. In our study, however, we observed that the in vitro rate of formation of the GSH conjugate was lower in the resistant species. Because we added the reduced GSH externally and it readily reacts with the reactive metabolites of PAs,23,41 the lower rate of mono-GSHDHP formation may explain the slower rate of reactive metabolite formation, but it does not explain the inefficiency of the sheep or rabbits to undertake GSH conjugation. In conclusion, lasiocarpine was extensively metabolized by the liver microsomes of all species examined except rabbits. There were a total of 12 oxidative metabolites of lasiocarpine, and these could be grouped into the dehydrogenation, ester bond cleavage, demethylation, N-oxidation, or hydroxylation metabolic pathways. Human liver microsomes displayed some distinctive features, such as a relatively high rate of reactive metabolite formation and low rate of demethylation metabolite formation. Furthermore, a high rate of formation of a new class of dehydrogenation metabolites (M4−M7) was also observed with human liver microsomes, indicating that humans may be more prone to lasiocarpine-induced acute toxicity than many other species. On the other hand, liver microsomes from resistant species (i.e., rabbits and sheep) produced lower levels of the reactive metabolites, as measured by the amount of the GSH conjugate as well as M1.

weight (311 g/mol), although M4 eluted over 2.5 min before M5. Similarly, M6 eluted over 3 min before M7 despite having the same molecular weight (325 g/mol). Moreover, M4 and M6, but not M5 and M7, were unstable in the presence of reduced GSH. This might indicate that the only difference between M4 and M6 or M5 and M7 was the presence of an O− CH3 group in the necic acid moiety of M6 and M7 (Scheme 1). In other words, M4 and M5 might be further oxidation products of M9 (i.e., the demethylation metabolite), whereas M6 and M7 were formed from lasiocarpine. On the basis of the ESI-MS/MS data, we propose that these metabolites might have a dehydrogenated necine base containing a ketone group at the C7 atom (Scheme 1). In fact, a metabolite with a similar molecular structure (i.e., danaidone) has been detected in insects fed PAs.33 Moreover, after oxidation of heliotrine with KMnO4, an earlier study reported the formation of 7-oxo derivatives of dehydroheliotrine,34 which were hydrolyzed to the keto-alcohol (Scheme S1).35 However, the elucidation of the molecular structure and the identification of the mechanisms of their formation and toxicological significance of these new metabolites of lasiocarpine warrant future investigation. DHP, which was reported as one of the major metabolites of lasiocarpine via the dehydrogenation pathway,5 was not detected in this study. Similarly, Segall et al. (1984) also reported that they could not detect DHP after in vitro mouse liver microsomal oxidation of senecionine.36 There are major species-specific differences in susceptibility to PA intoxication; these may be mediated by several biochemical factors such as CYP-mediated capacity of bioactivation, efficiency of N-oxidation, and carboxylesterasemediated hydrolysis as well as the level of cellular GSH.14,15,18,37,38 We examined the species-dependent variations in the metabolic detoxification and bioactivation of lasiocarpine. It seemed that M9 was the most abundant metabolite and the major detoxification product of lasiocarpine in all of the species studied. However, the relative abundance of M9 was much lower with human liver microsomes compared to that with liver microsomes from other species. Thus, humans maybe more prone to lasiocarpine-induced toxicity than the other species included in this study. Structurally, M9 qualifies as a genuine heliotridine-type PA, and it can be further bioactivated to reactive intermediates like any other PA. Therefore, classification of M9 as a detoxification metabolite was only based on the increased hydrophilicity of this metabolite, which may facilitate its excretion. For instance, in fast metabolizers, such as HL22, the level of M9 decreased sharply after 30 min of incubation, perhaps indicating further oxidation of the metabolite (Figure S8). Likewise, Odemethylation of heliotrine produced heliotridine trachelanthate, a less toxic metabolite.33 Dealkylation reactions are among the common reaction of CYPs.39 We also measured the species-dependent differences in the bioactivation capacity, i.e., the rate of the formation of M1 ((3H-pyrrolizin-7-yl)methanol) and the GSH conjugate, of the toxic metabolites of lasiocarpine. These metabolites are considered to be indirect measures of toxic pyrrolic esters such as lasiocarpine.23,40 Thus, a higher rate of formation of M1 or the GSH conjugate in vitro may indicate a higher capacity for lasiocarpine to be metabolically bioactivated by liver microsomes from susceptible species in comparison to that from resistant species such as rabbit and sheep. Earlier studies also support our findings that the resistance of sheep to senecionine toxicity can be attributed to their lower rate of pyrrolic

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.5b00253. ESI-MS/MS spectra of M1, M3, M8, M11, and M12 (Figure S1); LC-MS chromatogram of (3H-pyrrolizin-7yl)methanol) M1 and its minor isomer M2 (Figure S2); metabolism of M9 to M3 (Figure S3); confirmation of molecular structure of M9 (Figure S4); ESI-MS/MS spectra of M9 and echimidine (Figure S5); ESI-MS/MS spectra of lasiocarpine N-oxide (Figure S6); ESI-MS/MS fragment ions of the EC cell oxidation products of retrorsine (m/z 366 and m/z 384) (Figure S7); timedependent formation of M9 (m/z 398) in the liver microsomes of different species in the presence of NADPH (Figure S8); and preparation of 7-oxo derivatives of dehydroheliotrine (Scheme S1) (PDF).

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AUTHOR INFORMATION

Corresponding Author

*E-mail: markku.pasanen@uef.fi. Funding

The current study was funded by Academy of Finland (grant 137589), FPDP-Toxicology section, Finnish Cultural Foundation and VTR K59781. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank H. Jaatinen and Miia Reponen for technical assistance during in vitro metabolism studies and LC-MS analysis. I

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ABBREVIATION CYP, cytochrome P450 enzymes; DHP, dehydroretronecine; GSH, glutathione; IPCS, International Program on Chemical Safety; LC-MS, liquid chromatography/mass spectrometry; PAs, 1,2-dehydropyrrolizidine alkaloids; Q-ToF MS, time-offlight mass spectrometer

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K

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