7-Glutathione Pyrrole Adduct: A Potential DNA Reactive Metabolite of

Article pubs.acs.org/crt

7‑Glutathione Pyrrole Adduct: A Potential DNA Reactive Metabolite of Pyrrolizidine Alkaloids Qingsu Xia,† Liang Ma,† Xiaobo He,† Lining Cai,‡ and Peter P. Fu*,† †

National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, Arkansas 72079, United States Biotranex LLC, Monmouth Junction, New Jersey 08852, United States

‡

S Supporting Information *

ABSTRACT: Pyrrolizidine alkaloid (PA)-containing plants are the most common poisonous plants affecting livestock, wildlife, and humans. PAs require metabolic activation to form pyrrolic metabolites to exert cytotoxicity and tumorigenicity. We previously determined that metabolism of tumorigenic PAs produced four DNA adducts, designated as DHPdG-3, DHP-dG-4, DHP-dA-3, and DHP-dA-4, that are responsible for liver tumor initiation. 7-Glutathione-(±)-6,7-dihydro-1-hydroxymethyl5H-pyrrolizine (7-GS-DHP), formed in vivo and in vitro, and 7,9-di-GSDHP, formed in vitro, are both considered detoxified metabolites. However, in this study we determined that incubation of 7-GS-DHP with 2′-deoxyguanosine (dG) and 2′-deoxyadenosine (dA) yields DHPdG-3, DHP-dG-4, DHP-dA-3, and DHP-dA-4 adducts as well as the reactive metabolite DHP. Furthermore, reaction of 7-GS-DHP with calf thymus DNA in aqueous solution at 37 °C for 4, 8, 16, 24, 48, or 72 h, followed by enzymatic hydrolysis yielded DHP-dG-3, DHP-dG-4, DHP-dA-3, and DHP-dA-4 adducts. Under our current experimental conditions, DHP-dA-3 and DHP-dA-4 adducts were formed in a trace amount from the reaction of 7,9-di-GS-DHP with dA. No DHP-dG-3 or DHP-dG-4 adducts were detected from the reaction of 7,9-di-GS-DHP with dG. This study represents the first report that the 7-GS-DHP adduct can be a potential reactive metabolite of PAs leading to DNA adduct formation.

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4 (Figure 2).7,9 The level of DNA adduct formation correlated with the liver tumor potency of the PAs tested. These DNA adducts were not formed in control rats or in rats dosed with PAs that do not induce liver tumors.7 Thus, these DHP-dG and DHP-dA adducts are biomarkers of PA exposure and probable biomarkers of PA-induced liver tumors.7 In addition to the formation of DHP-dG-3, DHP-dG-4, DHP-dA-3, and DHP-dA-4 adducts, rat liver microsomal metabolism of riddelliine and senkirkine in the presence of calf thymus DNA also generated two sets of epimers, designated as DHP-dG-1, DHP-dG-2, DHP-dA-1, and DHPdA-2, as minor DNA adduct products (Figure 2).7,9,10 These results suggest that DHP-dG-1, DHP-dG-2, DHP-dA-1, and DHP-dA-2 adducts may also be generated in vivo. It has been reported that dehydro-PAs react with glutathione (GSH) to form 7-glutathionyl-(±)-6,7-dihydro-1-hydroxymethyl-5H-pyrrolizine (7-GS-DHP) in vitro and in vivo11−16 and 7,9di-GS-DHP in vitro.13,15,16 Both 7-GS-DHP and 7,9-di-GSDHP have been considered detoxified metabolites (Figure 1).1,2,17−22 In this study, we determined that reaction of 7-GSDHP with 2′-deoxyguanosine (dG), 2′-deoxyadenosine (dA), and calf thymus DNA generates DHP-dG adducts and/or

INTRODUCTION Pyrrolizidine alkaloids (PAs) and their N-oxide derivatives are common phytochemical constituents of hundreds of plant species widely distributed in many geographical regions of the world.1,2 There are 660 PAs and PA N-oxides present in over 6,000 plants, and more than half of the PAs identified are hepatotoxic. PAs and PA-containing plant extracts have also been found to induce tumors in experimental animals.1,2 PAcontaining plants are probably the most common poisonous plants affecting livestock, wildlife, and humans. PAs require metabolic activation to generate dehydro-PAs as the primary pyrrolic metabolites that are rapidly hydrolyzed at the C7- and C9-positions to produce (±)-6,7-dihydro-7hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP), which is less reactive (tumorigenic) than dehydro-PAs (Figure 1). 1,2 Dehydro-PAs are capable of binding to cellular proteins and DNA in the liver in vivo to produce protein-DHP adducts and DNA-DHP adducts, resulting in cytotoxicity, genotoxicity, and tumorigenicity.1−8 We recently reported that rats orally gavaged with seven individual hepatotumorigenic PAs and one PA N-oxide developed liver tumors through a general genotoxic mechanism mediated by a set of four DHP-derived DNA adducts, i.e., a pair of epimers designated as DHP-dG-3 and DHP-dG-4, and another pair of epimers designated as DHP-dA-3 and DHP-dA© 2015 American Chemical Society

Received: October 14, 2014 Published: March 13, 2015 615

DOI: 10.1021/tx500417q Chem. Res. Toxicol. 2015, 28, 615−620

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Chemical Research in Toxicology

Figure 1. Metabolism pathways of PAs leading to detoxication and the formation of reactive primary pyrrolic dehydro-PA leading to hepatotoxicity and liver tumors. from Fisher Scientific (Pittsburgh, PA). AX 1-X8 anion exchange resin was purchased from Bio-Rad (Hercules, CA). DNA samples were extracted using Blood & Cell Culture DNA Isolation Kit (QIAGEN Inc., Valencia, CA) according to the manufacturer’s instructions. The concentration of the DNA was determined spectrophotometrically. Dehydromonocrotaline and DHR were synthesized by dehydrogenation of monocrotaline and retronecine in chloroform with o-chloranil, as previously described.8,9 All solvents used were HPLC grade. Synthesis of DHP-dG and DHP-dA Adducts. Following our previously published procedures,9,10 DHP-dG-1 and DHP-dG-2 adducts were synthesized from a reaction of DHR with dG. DHPdA-1 and DHP-dA-2 adducts were synthesized from a reaction of DHR with dA; DHP-dG-3 and DHP-dG-4 were synthesized from a reaction of dehydromonocrotaline with dG; and DHP-dA-3 and DHPdA-4 adducts were synthesized from a reaction of dehydromonocrotaline with dA. Isotopically labeled DHP-[15N5]dG and DHP[15N513C10]dA adducts, used as internal standards for DHP-dG and DHP-dA, were synthesized as previously described.7 Synthesis of 7-GS-DHP and 7,9-Di-GS-DHP Adducts. For the synthesis of 7-GS-DHP, GSH (13 mg, 42 μmol) in 70 μL of PBS (0.1M, pH, 7.4) was added to dehydromonocrotaline (20 mg, 62 μmol) in 1 mL of DMF. The reaction mixture was stirred at ambient temperature for 2 h and quenched with 10 mL of PBS, and the resulting 7-GS-DHP was purified by HPLC. The 7,9-Di-GS-DHP adduct was prepared under similar conditions with the exception that a 7-fold excess of GSH was used for the reaction. DHP-dG and DHP-dA Adduct Formation from the Reaction of 7-GS-DHP with dG and dA. One milliliter of the reaction mixture (containing 1 mM 7-GS-DHP and 2 mM dG in ammonium hydroxide solution at pH 8) was incubated at 37 °C with shaking. The reaction was monitored by HPLC at 0, 1, 3, and 5 days to detect the formation of DHP-dG and DHP. For HPLC analysis, a Waters Alliance HPLC system (Milford, MA) consisting of Waters 2695 Separations Module and a Waters 2996 photodiode array detector was used. HPLC conditions: Phenomenex Luna C18 (2) column, 4.6 × 250 mm, (Phenomenex Inc. CA), monitored at 256 nm (for DHP-dG) and 220 nm (for DHP), respectively, and isocratic elution with 12% acetonitrile in water at a flow rate of 1 mL/min. Formation of DHP-dA adducts from the reaction of 7-GS-DHP and dA was similarly conducted, with the exception that the reaction was assessed only after 7 days. HPLC conditions: monitored at 269 nm

Figure 2. Structures of DHP-dG-1, DHP-dG-2, DHP-dG-3, DHP-dG4, DHP-dA-1, DHP-dA-2, DHP-dA-3, and DHP-dA-4 adducts.

DHP-dA adducts. These results suggest that the 7-GS-DHP adduct can be a potential DNA reactive metabolite of PAs.

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EXPERIMENTAL PROCEDURES

Caution: Monocrotaline, dehydromonocrotaline, and dehydroretronecine (DHR) are carcinogenic in laboratory animals. They should be handled with extreme care, using proper personal protective equipment and a well-ventilated hood. Chemicals. Monocrotaline, o-chloranil, calf thymus DNA (sodium salt, type I), nuclease P1, micrococcal nuclease (MN), spleen phosphodiesterase (SPD), and PBS were purchased from SigmaAldrich (St. Louis, MO). Dimethylformamide (DMF), acetonitrile, potassium carbonate, chloroform, and diethyl ether were obtained 616

DOI: 10.1021/tx500417q Chem. Res. Toxicol. 2015, 28, 615−620

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Chemical Research in Toxicology

Figure 3. HPLC profiles of the reaction of 7-GS-DHP with dG: (left panel) detection of DHP formation monitored at 220 nm and (right panel) detection of DHP-dG adducts monitored at 256 nm. For reaction conditions, see Experimental Procedures. the DHP-[15N5 13C10]dA internal standards at the [M + H]+ m/z 402 to m/z 263 transition. Samples were quantified by comparing the areas of the unlabeled chromatogram peaks to those of the corresponding labeled internal standard chromatogram peaks. Data acquisition and reprocessing were performed using the Thermo Xcalibur 2.0 SR2 software. Standard Characterization and Calibration Curve. Standard curves were generated by plotting the amounts of standard compounds against peak area. Each sample was tested at 10 μL injection volume containing 100 fmol DHP-[15N5]dG and 25 fmol DHP-[15N5,13C10]dA as internal standards. The calibration curves were linear over the concentration range of 1−400 fmol for DHP-dG and 2−200 fmol for DHP-dA. The best linear fit and least variability for the calibration curve were achieved with a weighting factor of 1/X, with the correlation coefficients (r2) for all analyses above 0.99.

(for DHP-dA) and 220 nm (for DHP), and isocratic elution with 15% acetonitrile in water at a flow rate of 1 mL/min. Reaction of 7,9-Di-GS-DHP with dG and dA. A solution of 7,9di-GS-DHP (1 mM) and dG (2 mM) or dA (2 mM) in 1 mL of deionized water or ammonium hydroxide solution at pH 8 was kept at 37 °C with shaking for 7 days, respectively. The resulting reaction mixtures were analyzed by LC/MS/MS. Reaction of 7-GS-DHP with Calf Thymus DNA. A solution of 7GS-DHP (0.5 mM) and calf thymus DNA (1 mg) in 1 mL of ammonium hydroxide solution at pH 8 was stirred at 37 °C for 4, 8, 16, 24, 48, or 72 h, respectively. After the reaction, 100 μL of each reaction mixture (100 μg DNA) was enzymatically hydrolyzed to nucleotides with micrococcal nuclease, spleen phosphodiesterase, and nuclease P1 as previously described.10 Reactions at each time point were conducted three times (n = 3). Quantitation of DHP-dG and DHP-dA Adducts by LC/MS/MS Analysis. Lipid Chromatography. A Finnigan Surveyor HPLC system was coupled with a TSQ mass spectrometer. The samples were loaded onto a reverse phase column (ACE 3 C18, 4.6 mm × 150 mm, 3 μm, MAC-MOD Analytical, Chadds Ford, PA) with a gradient of methanol and water (containing 2 mM ammonium acetate, pH 5) with a flow rate of 0.3 mL/min. The gradient began with 15% methanol for 5 min, followed by a linear gradient up to 65% methanol over the next 35 min, then methanol was increased to 95% in 2 min. After holding 95% methanol for 6 min, the gradient was reset to 15% methanol in 2 min. The column was equilibrated for 18 min before the next injection. The total run time for one injection was 68 min. The samples were maintained at 5 °C in the autosampler during the entire analysis. Mass Spectrometry. A TSQ Quantum Ultra Triple Stage Quadrupole MS/MS System (ThermoFinnigan, San Jose, CA, USA) equipped with an atmospheric pressure ionization (API) electrospray (ESI) interface was used to perform the MS−MS analyses. For the DHP-DNA adducts assay, the spray voltage was 3000 V, vaporizer temperature was 400 °C, capillary temperature was 280 °C, and tube lens offset was 76 V. Nitrogen pressures of the sheath and auxiliary gases were 30 and 5 (arbitrary units), respectively. The argon collision gas pressure was 1.5 mTorr, and the collision energy was 17 eV for DHP-dG and its internal standard, and 21 eV for DHP-dA and its internal standard. Positive ions were acquired in the selected reaction monitoring (SRM) mode (dwell time of 100 ms for each analyte and internal standard). The DHP-dG adducts were monitored at the [M + H]+ m/ z 403 to m/z 269 transition and the DHP-[15N5]dG internal standards at the [M + H]+ m/z 408 to m/z 274 transition. The DHP-dA adducts were monitored at the [M + H]+ m/z 387 to m/z 253 transition and

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RESULTS DHP-dA and DHP-dG Adducts Formed from the Reactions of 7-GS-DHP with dG and dA. The reaction of 7-GH-DHP with dG was monitored at 256 nm by HPLC at 0, 1, 3, and 5 days for the DHP-dG adduct formation. As shown in Figure 3, at day 1, DHP-dG-1 and DHP-dG-2 were formed; and at day 5, DHP-dG-3 and DHP-dG-4 were formed as minor adducts. The identity of DHP-dG products was confirmed by LC-MS/MS analysis and compared with synthetic standards. The reaction yields of DHP-dG-1 and DHP-dG-2 increased in a concentration and time dependent manner. The formation of DHP was monitored at 220 nm by HPLC (Figure 3) and confirmed with the HPLC retention time and mass of the authentic DHP. The yield of DHP was also increased in a time dependent manner. Reaction of 7-GS-DHP and dA in water for 7 days was similarly conducted. As shown in Figure 4, DHP-dA-1, DHPdA-2, DHP-dA-3, and DHP-dA-4 were formed. On the basis of the previous assumption that the molar extinction coefficients of DHP-dA-1, DHP-dA-2, DHP-dA-3, and DHP-dA-4 are identical,9 the yields of DHP-dA-1 and DHP-dA-2 are higher than that of DHP-dA-3 and DHP-dA-4. Similar to the reaction of 7-GS-DHP with dG, DHP was also produced. Reactions of 7,9-di-GS-DHP with dA or dG for 7 days were similarly conducted. No DHP-dG or DHP-dA adducts were detected by HPLC; by LC-ES-MS/MS analysis, DHP-dA-3 and DHP-dA-4 adducts were detected in a trace amount. 617

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results clearly indicate that all four DHP-dG and four DHP-dA adducts were formed. As shown in Figure 6, with the exception of the reaction yields of DHP-dG adducts formed at 72 h, the yields of DHPdG and DHP-dA adducts increased in a time dependent manner (Figure 6A and B). The levels of DHP-dG-3/4 adducts are significantly higher than those of DHP-dG-1/2 adducts. Similarly, the levels of DHP-dA-3/4 adducts are higher than those of DHP-dA-1/2 adducts (Figure 6A and B). The formation of DHP was also determined by LC-ES-MS/ MS analysis (Figure 6C) and confirmed by comparison with the LC-ES-MS/MS profile of authentic DHP. The yields of DHP increased with time up to 48 h (Figure 6C). Similar to DHP-dG-3/4 (Figure 6A), the yield at 72 h was lower than that at 48 h (Figure 6C).

Figure 4. HPLC profile of the reaction of 7-GS-DHP with dA for 7 days. Detection of DHP-dA adducts monitored at 268 nm.

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LC-ES-MS/MS Analysis of DHP-dG and DHP-dA Adducts Formed from a Reaction of 7-GS-DHP and Calf Thymus DNA. DHP-dG and DHP-dA adducts formed from a reaction of 7-GS-DHP with calf thymus DNA in ammonium hydroxide solution at pH 8 for 4, 8, 16, 24, 48, and 72 h were quantitated by LC-ES-MS/MS through selected reaction monitoring (SRM) with the use of DHP-[15N5]dG-3/ 4 and [15N5 13C10]dA-3/4 as internal standards. Representative SRM measurements for DHP-dG and DHP-dA adducts formed from the reaction of 7-GS-DHP with calf thymus DNA for 16 and 72 h, respectively, are shown in Figure 5. SRM chromatograms from reactions of 4, 8, 24, and 48 h are shown in Figures S1 and S2 in the Supporting Information. The

DISCUSSION It has been reported that metabolism of PAs in vivo produces reactive pyrrolic dehydro-PA metabolites that bind to cellular proteins and DNA to form protein-DHP adducts and DNADHP adducts leading to toxicity and tumorigenicity.2,7 The formation of 7-GS-DHP from the conjugation of dehydro-PAs with glutathione in vivo has long been considered a detoxication pathway.1,2 In this article, we demonstrate that 7-GS-DHP can react with calf thymus DNA, dG, and dA to form DNA adducts (Figures 3−6). These are the same DHP-dG and DHP-dA adducts that have been shown to be responsible for the induction of liver tumors in rats administered PAs.7,10 These

Figure 5. LC/MS SRM chromatograms of DHP-dG and DHP-dA adducts formed from the reaction of 7-GS-DHP with calf thymus DNA for 16 h (left panel) and 72 h (right panel). IS: DHP-[15N5]dG and DHP-[15N5,13C10]dA labeled internal standards. 618

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Figure 7. Proposed general metabolic activation of carcinogenic PAs leading to the formation of DHP-derived DNA adducts that potentially cause PA-induced liver tumors.

The formation of DHP-derived DNA adducts from a reaction of 7-GS-DHP with dG, dA, and calf thymus DNA can result from (i) the direct binding of 7-GS-DHP to dG, dA, and calf thymus DNA; (ii) the hydrolysis of 7-GS-DHP to DHP which subsequently binds to dG, dA, and calf thymus DNA; or (iii) a combination of both. At the present, it is not known which route predominates. The results presented in this present article illustrate that although formation of glutathione conjugates is generally considered a detoxification pathway, glutathione conjugates can sometimes be toxic and genotoxic metabolites. Guengerich and co-workers have demonstrated this for glutathione conjugates of dihaloalkanes.24,25 For example, 1,2-dibromoethane is metabolized to the reactive intermediate S-(2bromoethyl)glutathione (or the derived episulfonium ion) that binds to DNA to form the adduct (S-[2-(N7-guanyl)ethyl]glutathione). In contrast, DNA adduct formation from 7GS-DHP occurs through replacement of the GSH group by DNA (Figure 7). Whether 7-GS-9-DNA-DHP is formed in vivo remains to be determined.

Figure 6. Formation of (A) DHP-dG adducts, (B) DHP-dA adducts, and (C) DHP from the reaction of 7-GS-DHP with calf thymus DNA in aqueous medium at pH 8 and 37 °C for 4, 8, 16, 24, 48, and 72 h (n = 3).

results strongly suggest that 7-GS-DHP can react with cellular DNA in vivo to generate DHP-dG and DHP-dA adducts and thus initiate liver tumor formation. On the basis of our overall findings, we propose that toxic PAs possess three possible activated metabolites: dehydro-PA, DHP, and 7-GS-DHP (Figure 7). The role of 7-GS-DHP in PA-induced liver tumorigenicity is not yet known. Dehydro-PAs are highly unstable, having halflives of only 0.3−5.1 s in the aqueous medium.23 7-GS-DHP is more stable; it can potentially serve as a reservoir, being translocated to other organs to cause toxic effects in extrahepatic tissues, such as the lung. Thus, it will be important to evaluate the role of 7-GS-DHP in extra-hepatic toxicity. dG and dA preferentially attack at the C7 position of 7-GSDHP, resulting in the removal of the GSH group at the C7position (Figure 3 and Figure 4). In contrast, calf thymus DNA predominantly attacks the C9-position of 7-GS-DHP, and the hydroxyl group at the C9-position is the leaving group (Figures 5 and 6). The observed preference of calf thymus DNA binding at the C9-position is likely ascribed to steric hindrance since calf thymus DNA is much larger than dG and dA, and the C9position is less hindered than the C7-position.9

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ASSOCIATED CONTENT

S Supporting Information *

LC/MS SRM chromatograms of DHP-dG and DHP-dA adducts from different reaction times. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +1-870-543-7207. Fax: +1-870-543-7136. E-mail: peter. [email protected]. Funding

This research was supported in part by appointments (L.M. and X.H.) 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. 619

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Chemical Research in Toxicology Notes

evidence for the formation of a pyrrolic metabolite of an otonecinetype pyrrolizidine alkaloid. Drug Metab. Dispos. 26, 181−184. (14) Mattocks, A. R., Croswell, S., Jukes, R., and Huxtable, R. J. (1991) Identity of a biliary metabolite formed from monocrotaline in isolated, perfused rat liver. Toxicon 29, 409−415. (15) Reed, R. L., Miranda, C. L., Kedzierski, B., Henderson, M. C., and Buhler, D. R. (1992) Microsomal formation of a pyrrolic alcohol glutathione conjugate of the pyrrolizidine alkaloid senecionine. Xenobiotica 22, 1321−1327. (16) Tamta, H., Pawar, R. S., Wamer, W. G., Grundel, E., Krynitsky, A. J., and Rader, J. I. (2012) Comparison of metabolism-mediated effects of pyrrolizidine alkaloids in a HepG2/C3A cell-S9 coincubation system and quantification of their glutathione conjugates. Xenobiotica 42, 1038−1048. (17) Lin, G., Cui, Y. Y., and Hawes, E. M. (2000) Characterization of rat liver microsomal metabolites of clivorine, an hepatotoxic otonecine-type pyrrolizidine alkaloid. Drug Metab. Dispos. 28, 1475− 1483. (18) Lin, G., Wang, J. Y., Li, N., Li, M., Gao, H., Ji, Y., Zhang, F., Wang, H., Zhou, Y., Ye, Y., Xu, H. X., and Zheng, J. (2011) Hepatic sinusoidal obstruction syndrome associated with consumption of Gynura segetum. J. Hepatol. 54, 666−673. (19) White, I. N. (1976) The role of liver glutathione in the acute toxicity of retrorsine to rats. Chem.-Biol. Interact. 13, 333−342. (20) Yan, C. C., and Huxtable, R. J. (1995) The effect of the pyrrolizidine alkaloids, monocrotaline and trichodesmine, on tissue pyrrole binding and glutathione metabolism in the rat. Toxicon 33, 627−634. (21) Yan, C. C., and Huxtable, R. J. (1995) Relationship between glutathione concentration and metabolism of the pyrrolizidine alkaloid, monocrotaline, in the isolated, perfused liver. Toxicol. Appl. Pharmacol. 130, 132−139. (22) Yan, C. C., Cooper, R. A., and Huxtable, R. J. (1995) The comparative metabolism of the four pyrrolizidine alkaloids, seneciphylline, retrorsine, monocrotaline, and trichodesmine in the isolated, perfused rat liver. Toxicol. Appl. Pharmacol. 133, 277−284. (23) Cooper, R. A., and Huxtable, R. J. (1999) The relationship between reactivity of metabolites of pyrrolizidine alkaloids and extrahepatic toxicity. Proc. West Pharmacol. Soc. 42, 13−16. (24) Guengerich, F. P. (2003) Review: Activation of dihaloalkanes by thiol-dependent mechanisms. Biochem. Mol. Biol. Rep. 36, 20−27. (25) Peterson, L. A., Harris, T. M., and Guengerich, F. P. (1988) Evidence for an episulfonium ion intermediate in the formation of S[2-(N7-guanyl) ethyl] glutathione in DNA. J. Am. Chem. Soc. 110, 3284−3291.

This article is not an official U.S. Food and Drug Administration (FDA) guidance or policy statement. No official support or endorsement by the U.S. FDA is intended or should be inferred. The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Drs. Barry Delclos and Frederick A. Beland for critical review.

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ABBREVIATIONS DHR, dehydroretronecine or (−)-R-6,7-dihydro-7-hydroxy-1hydroxymethyl-5H-pyrrolizine; DHP, (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine; 7-GS-DHP, 7-glutathione-DHP; 7,9-di-GS-DHP, 7,9-diglutathione-DHP; LC-ESMS/MS, high-performance liquid chromatography−electrospray ionization−tandem mass spectrometry; SRM, selected reaction monitoring; NCTR, National Center for Toxicological Research

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REFERENCES

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