Hemoglobin Adduct Levels in Rat and Mouse ... - ACS Publications

Chem. Res. Toxicol. 2004, 17, 785-794

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Hemoglobin Adduct Levels in Rat and Mouse Treated with 1,2:3,4-Diepoxybutane Charlotta Fred,† Antti Kautiainen,‡ Ioannis Athanassiadis,† and Margareta To¨rnqvist*,† Department of Environmental Chemistry, Stockholm University, SE-106 91 Stockholm, Sweden, and Preclinical R&D, Biovitrum AB, SE-112 76 Stockholm, Sweden Received October 21, 2003

For cancer risk assessment of 1,3-butadiene from rodent cancer test data, the in vivo doses of formed 1,2:3,4-diepoxybutane (DEB) should be known. In vivo doses of DEB were measured through a specific reaction product with hemoglobin (Hb), a ring-closed adduct, N,N-(2,3dihydroxy-1,4-butadiyl)valine (Pyr-Val), to N-terminal valines. An analytical method based on tryptic digestion of Hb and quantification of Pyr-modified heptapeptides by LC-MS/MS has been further developed and applied in vivo to DEB-treated rats. Furthermore, N-(2,3,4trihydroxybutyl)valine adducts (THB-Val) to the N-terminal valine in Hb were measured in rats and mice treated with DEB and in a complementary experiment with 1,2-epoxy-3,4butanediol (EBdiol), using a modified Edman degradation method and GC-MS/MS. In vitro reactions of hemolysate with DEB and EBdiol were used to measure reaction rates for adduct formation needed for calculation of doses and rates elimination in vivo. The results showed that the level of the Pyr-Val adduct per administered dose of DEB was approximately the same in rats as had earlier been observed in mice [Kautiainen et al. (2000) Rapid Commun. Mass Spectrom. 14, 1848-1853]. Levels of the THB-Val adduct after DEB treatment were 3-4 times higher in rat than in mouse, probably reflecting an enhanced hydrolysis of DEB to EBdiol catalyzed by epoxide hydrolase. After EBdiol treatment, the THB-Val adduct levels were about the same in rat and mouse. Calculations from in vitro data show that the Pyr-Val adduct is a relevant monitor for the in vivo dose of DEB and that THB-Val primarily reflects doses to EBdiol. The calculated rates of formation of adducts and rates of elimination agree with expectations. Procedures for quantification of Hb adducts as modified peptides as well as preparation and characterization of peptide standards have been evaluated.

Introduction 1,3-Butadiene (BD), which is a common chemical in the polymer industry, is considered by the U.S. EPA to be a human carcinogen (1). The human lifetime cancer risk for chronic exposure to BD has recently been estimated to be 0.08 per ppm by the U.S. EPA (1) based on increased leukaemia risks observed in occupationally exposed workers (2). This risk assessment is, however, still very uncertain. BD is a multisite rodent carcinogen; mice are more sensitive than rats. Chronic (2 years) inhalation exposure to concentrations of g1000 ppm BD is needed to provoke a significant increase in the tumor incidence in rats (3) while mice develop tumors at and above 6.25 ppm BD (4). Because of the large interspecies difference in response to BD, it is difficult to estimate the risks to humans by extrapolation from cancer test data. This difference in BD-induced carcinogenicity has been suggested to be due to species-related differences in the enzymatic production and detoxication of reactive metabolites (5, 6). BD is oxidized by cytochrome P450 enzymes, notably CYP 2E1, to the monoepoxide, EB,1 and its diepoxide, * To whom correspondence should be addressed. Tel: +46-8-16 37 69. Fax: +46-8-16 39 79. E-mail: [email protected]. † Stockholm University. ‡ Biovitrum AB.

DEB (7). Each of these metabolites can be further metabolized to EBdiol (7, 8). The formation and the further oxidation or hydrolysis of the reactive epoxides of BD are described in Figure 1. DEB is the most genotoxic epoxide metabolite of BD and is about 100 times more mutagenic than the monofunctional epoxide metabolites EB and EBdiol (9). This is assumed to be due to its ability to form cross-links in DNA (10-12). In cancer risk assessment, it is important to know the in vivo dose (i.e., concentration × time) of the genotoxic intermediate(s) (13). In vivo and in vitro measurements of concentrations of epoxides in blood have shown that as compared to rats, mice oxidize BD at a much higher rate and exhibit a distinctly higher ratio of activation to detoxication (14-17). One way of studying differences in metabolism and doses of epoxide metabolites between species, including man, would be to measure levels of Hb adducts in exposed humans and animals (18). Adducts to the N-terminal amino acid (valine) in mouse, rat, and human Hb from the monofunctional metabolites of BD are analyzable by 1 Abbreviations: DEB, 1,2:3,4-diepoxybutane; EBdiol, 1,2-epoxy-3,4butanediol; EB, 1,2-epoxy-3-butene; EH, epoxide hydrolase; Hb, hemoglobin; Pyr-Val, N,N-(2,3-dihydroxy-1,4-butadiyl)valine; THB-Val, N-(2,3,4-trihydroxybutyl)valine; (2H5)DHP-Val, N-(2,3-dihydroxy(2H5)propyl)valine; PFPITC, pentafluorophenyl isothiocyanate; PFPTH, pentafluorophenylthiohydantoin; ESI, electrospray ionization; CID, collision-induced dissociation; SRM, selected reaction monitoring.

10.1021/tx034214g CCC: $27.50 © 2004 American Chemical Society Published on Web 05/08/2004

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Fred et al.

coupling/cleavage reaction (Figure 2) (20). Adducts to N-terminal valine in Hb, 2-hydroxy-3-butenyl and THBVal, have been measured in humans and experimental animals exposed to BD or its different epoxide metabolites (Figure 2) (19, 21, 22). Preliminary kinetic studies indicate that the ring closure to Pyr-Val of the initially formed 3,4-epoxy-2-hydroxybutyl adduct from DEB is relatively fast and that the formation of the THB adduct through hydrolysis may be a minor pathway (20), whereas Pyr-Val would be a relevant dose monitor for DEB. For the analysis of the ring-closed DEB adduct to the N-terminal valines in Hb (Pyr-Val), an analytical method based on tryptic digestion of globin and analysis of modified peptides has been developed and applied to mouse Hb (23). The formed N-terminal pyrrolidine heptapeptide (Pyr-heptapeptide) of the R-chain from trypsination of Hb is enriched by HPLC and analyzed by LCMS/MS. With this method, the Pyr-Val adduct was quantified in mice exposed to DEB (23).

Figure 1. Genotoxic metabolites formed from metabolism of BD (1). The metabolites most relevant with regard to genotoxicity are considered to be EB, DEB, and EBdiol.

a modified Edman degradation for detachment of alkylated N-terminal valines (19-21). However, this method is not applicable to adducts from the bifunctional metabolite DEB because of the formation of a ring-closed adduct, a pyrrolidine (Pyr-Val), which blocks the Edman

This paper describes further development and application of this method according to the following: (i) Preparation of standard peptides and evaluation of the applicability of the method for measurement of Hb adducts from DEB (Pyr-Val) as tryptic N-terminal peptides by LC-MS/MS to rat Hb; (ii) measurement of PyrVal adduct levels in DEB-treated rats; (iii) evaluation of the relevance of the Pyr-Val adduct for DEB exposure through complementary measurement of THB-Val adducts after treatment in vitro and in vivo with DEB and EBdiol; (iv) evaluation of detoxication rate of DEB in rat and comparison with mouse; and (v) evaluation of the procedure for measurement of protein adducts as modified peptides using LC-MS/MS.

Figure 2. Description of the two different methods used for analysis of adducts formed from reaction of DEB and EBdiol with Hb.

Biomonitoring of 1,3-Butadiene Metabolites

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Table 1. Modified Peptides/Hb from in Vitro and in Vivo Treatment with racDEB Standards in vitro-modified peptides

Pyr-modified peptide

THB-modified peptide

rat: heptapeptide corresponding to N-terminal R-chain in rat Hb treated in vitro with DEB

identification: LC-MS degree of alkylation: HPLC-UV application: standard for tuning of instruments

identification: LC-MS degree of alkylation: HPLC-UV application: calibration standard for quantification of THB adducts in mouse and rat

rat: (2H8)octapeptide corresponding to deuterium-substituted Val in N-terminal R-chain in rat Hb treated in vitro with DEB

identification: LC-MS degree of alkylation: HPLC-UV application: internal standard for quantification of Pyr adducts in rats (in vitro and in vivo)

identification: LC-MS degree of alkylation: HPLC-UV

mouse: heptapeptide corresponding to N-terminal R-chain in mouse Hb treated in vitro with DEB

identification: LC-MS degree of alkylation: HPLC-UV application: standard for tuning of instruments


mouse: (2H8)octapeptide corresponding to deuterium-substituted Val in N-terminal R-chain in mouse Hb treated in vitro with DEB

identification: LC-MS degree of alkylation: amino acid analysisa and HPLC-UV application: internal standard for quantification of Pyr adducts in mice (in vitro and in vivo)


Hb Samples in vitro/in vivo-modified Hb rat and mouse: Hb from in vitro treatment with DEB

Pyr-modified peptide preparation: trypsination and isolation of the respective R-chain N-terminal heptapeptide identification: LC-MS quantification: LC-MS/MS application: reaction kinetics

rat and mouse: Hb from in vitro treatment with EBdiol

rat and mouse: Hb from in vivo treatment with DEB

quantification: GC-MS/MS using the N-alkyl Edman method (see Figure 2) application: reaction kinetics preparation: trypsination and isolation of the respective R-chain N-terminal heptapeptidea quantification: LC-MS/MSa application: in vivo dose monitoring of DEB

rat and mouse: Hb from in vivo treatment with EBdiol

a

THB-modified valine identification of THB-modified peptides: LC-MS

quantification: GC-MS/MS using the N-alkyl Edman method (see Figure 2) application: in vivo dose monitoring of EBdiol quantification: GC-MS/MS using the N-alkyl Edman method (see Figure 2) application: in vivo dose monitoring of EBdiol

Mouse data earlier published by Kautiainen et al. (23).

Experimental Procedures Caution: DEB, EBdiol, and PFPITC are hazardous and should be handled carefully and always used in a hood (27). DEB and EBdiol are destroyed with 1 M H2SO4(aq). PFPITC is destroyed with NH3 in ethanol (1:1). Chemicals. DEB (97%) was obtained from Aldrich Chemical Co. (Milwaukee, WI), and EBdiol was earlier synthesized (24). Heptapeptide corresponding to the R-chain of mouse and rat globin sequences 1-7, Val-Leu-Ser-Gly-Glu-Asp-Lys and ValLeu-Ser-Ala-Asp-Asp-Lys, respectively, were from Neosystem Laboratoire (Strasbourg, France). The corresponding (2H8)octapeptides, (2H8)Val-Leu-Ser-Gly-Glu-Asp-Lys-Ser and (2H8)Val-Leu-Ser-Ala-Asp-Asp-Lys-Thr from mouse and rat, respectively, were also obtained from Neosystem Laboratoire. The purity of the peptides was >95% (net weight 64-73%). Acetic acid anhydride, triethylamine (>99%), and myoglobin from horse skeletal muscle (95-100%) were obtained from Sigma (Sigma-Aldrich Co., Steinheim, Germany). Trypsin (TPCKtreated) from bovine pancreas and PFPITC [purum, purified on

a Sep-Pak silica column (25)] were obtained from Fluka (Buchs, Switzerland). A globin modified at the N-terminal valine with 2,3-dihydroxy(2H5)propyl [(2H5)DHP-globin] was earlier synthesized (26). All other chemicals and solvents were of analytical grade. Glassware was silanized with Repel-Silane, a 2% (w/v) dimethyldichlorosilane solution in 1,1,1-trichloroethane from LKB-Produkter AB (Bromma, Sweden). Preparation of Peptide Standards. For use as calibration standards, internal standards, and also as standards for tuning of the analytical equipment, modified peptides were prepared (summarized in the Standards section of Table 1). The in vitro alkylation of hepta- and (2H8)octapeptides with DEB gives rise to both Pyr and THB modifications. The degree of these modifications was determined by HPLC-UV analysis (220 nm) using a concentration-response curve for the pure hepta- and (2H8)octapeptide according to Fred et al. (unpublished results).2 For chromatographic conditions, see Pyr-Val Adduct Analysis. The in vitro alkylation of peptides was done by addition of DEB (10 µL, 1 M) to an aqueous solution of hepta- or (2H8)octapeptide (0.1 mL, ca. 40 mM, pH 8). The concentration of

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Fred et al. The globin was isolated by addition of 2-propanol/HCl to the hemolysate and precipitated by dropwise addition of cold ethyl acetate (28). The globin was washed twice with ethyl acetate and once with pentane and dried overnight and then stored at -20 °C until further workup and analysis. In vitro-alkylated Hb was obtained through DEB and EBdiol incubations of hemolysed erythrocytes from untreated mouse and rat. Hemolysate in phosphate buffer (50 mM, pH 7.2) was incubated for 1-3 h at room temperature with 1-50 mM DEB or EBdiol. The globin was isolated as described above.

Figure 3. HPLC analysis of in vitro-modified heptapeptide, corresponding to the tryptic N-terminal heptapeptide in the R-chain of rat Hb. The more polar THB-modified heptapeptide elutes as a double peak (stereoisomers) around 17.5 min. The Pyr-modified heptapeptide eluting at 19.0 min also separates as a double peak if a slower gradient is applied. The mixture of unmodified and modified heptapeptides has been used as a standard (see Table 1). the peptides and the pH of the reaction solution were adjusted in order to increase the reactivity of valine and to minimize the alkylation of lysine and also the cross-linking of peptides (20). The solution was incubated at 37 °C for 72 h. The reaction products were characterized by LC-ESI-MS (see below). Mixtures of unmodified peptide with Pyr- and THB-modified peptides were used as peptide standards (Figure 3 and legend). The in vitro-modified heptapeptides were used as standards for the analysis of Pyr adduct in digested globins from Hb samples for determination of the retention time of Pyr-heptapeptide from mouse and rat in HPLC analysis and for tuning of the LC-MS/MS for quantification of Pyr adduct levels. Furthermore, the modified heptapeptide from rat was used as a calibration standard for quantitative measurement of THB adduct levels by the N-alkyl Edman method. The in vitro-modified (2H8)octapeptides were used as internal standards for Pyr adduct analysis in mouse and rat and added to the globin sample before digestion. It was cleaved to the modified (2H8)heptapeptide and was therefore expected to adjust for possible variations in yield during protein digestion. Treatment with DEB and EBdiol in Vivo and in Vitro and Isolation of Globin Samples. Male Fisher 344 rats and Sprague-Dawley rats (7 weeks of age) were obtained from Charles River Sverige AB (Uppsala, Sweden). Three rats (Fisher 344) per administered dose (100, 200, and 300 µmol/kg) were used and given a racemic mixture of DEB [(2R,3R)- and (2S,3S)DEB] dissolved in saline by i.p. injection. Male C57/Black mice (8 weeks of age) were obtained from the B & K-Universal AB (Sollentuna, Sweden). Five mice per administered dose (150 and 300 µmol/kg) were used and given an i.p. injection of DEB dissolved in saline. Two untreated rats (Fisher 344) and five untreated mice kept under the same conditions served as controls. In a complementary experiment, EBdiol was administered to one male rat (Sprague-Dawley) and two male mice (C57/ Black) per administered dose (230 and 460 µmol/kg) by i.p. injection. (Blood samples from the mice treated with the same dose of EBdiol were pooled.) After 24 h, the animals were sacrificed and blood was collected in heparinized tubes for Hb adduct measurement. Before sacrifice, the rats were anaesthetized with carbon dioxide and the mice were anaesthetized with metofan. The animal experiments were performed in agreement with the ethical approval N45/99 (Swedish National Board for Laboratory Animals). The number of animals was limited to the minimum required for evaluation of the method for adduct measurement. A few of the animals could also be used for a quantitative comparison of the species. Erythrocytes, separated from plasma and washed twice with PBS, were hemolysed by addition of distilled water (1.5 vol).

Commercially available myoglobin to be used as control globin was purified by a simpler method (29). The precipitation was done by dropwise addition of an aqueous solution (100 mg/mL) to a cold mixture of 1% HCl in acetone. The myoglobin was then washed twice with acetone and once with ether and dried overnight. Pyr-Val Adduct Analysis. 1. Enzymatic Degradation and Isolation of Modified Peptides. Pyr-Val adduct analysis in Hb was done in samples from DEB-treated rats (and control rats) and in samples from rat and mouse hemolysate treated in vitro with DEB. Plastic tubes and vials were used throughout the analysis. The procedure according to Kautiainen et al. (23) was applied. Globin samples (20 mg) were dissolved in water (2.4 mL), and Pyr-(2H8)octapeptide (80 µL, 2.1 nmol) was added as an internal standard (cf. Table 1). Sodium dodecyl sulfate (40 µL, 10% w/v) and ammonium hydrogen carbonate (1.4 mL, 0.5 M, pH 8.5) were added, and the samples were digested with trypsin at a substrate-to-enzyme ratio of 50:1 (w/w) for 16 h at 37 °C. Trypsin cleaves specifically at Lys and Arg, which leads to the formation of N-terminal heptapeptides from the R-chain in mouse and rat Hb. The digested globins were concentrated to ca. 300 µL by a vacuum centrifuge (Genvac SF50, Sales Development Ltd., Ipswich, U.K.), and the pH was lowered to ca. 2 by adding TFA. The samples were filtered using a 13 mm syringe filter GHP membrane (0.2 µm) (Pall Life Sciences, Lund, Sweden) and fractionated by semipreparative HPLC using an LKB 2150system, equipped with a controller, pumps, and an UV detector set at 220 nm. Reversed-phase chromatography was performed with a C-18 column (10 mm × 250 mm; Hichrom, Reading, U.K.). The gradient applied in the analysis was linearly changed from 5% acetonitrile(aq), 0.1% TFA, to 40% acetonitrile(aq), 0.1% TFA in 50 min. The flow rate was 3 mL/min. For each sample, the chromatographic fractions containing the Pyr-heptapeptide were pooled and concentrated by a centrifugal evaporator (Jouan RC 10-20, Jouan S. A., SaintHerblain, France) before quantification of Pyr-heptapeptides by LC-MS/MS. 2. LC-ESI-MS Analysis. Pyr-Val adduct levels in the modified peptides to be used as standards and in samples of globin from blood from the in vitro and in vivo treatments with DEB were determined by LC-MS/MS according to the following: Peptide standards (10 ng/µL, 5 µL) or fractionated globin samples (about 1/4 of the collected fraction, in 10 µL) were run on a C-18 column (1 mm × 100 mm, Xterra MS) using a Waters 2790 HPLC (Waters Chromatography, Milford, MA). The solvents consisted of 0.1% formic acid in 5% acetonitrile(aq) (solvent A) and of 0.1% formic acid in acetonitrile (solvent B), and the column was eluted with a linear gradient from 0 to 22% B in 25 min at a flow rate of 50 µL/min. The flow from the LC system was introduced into the ESI source of a Quattro Micro triple quadrupole mass spectrometer from Micromass (Manchester, U.K.) operated in the positive ionization mode. Nitrogen was used as the desolvation gas at a flow rate of 518 L/h. The ion source temperature of the mass spectrometer was 80 °C, the capillary voltage was 3.0 kV, and the cone voltage was 56 V. The mass spectrometer was used in full scan mode for characterization of the peptides and SRM for quantitative analysis. Data were acquired in the centroid mode. Acquisition and processing of data were performed using the commercial software MassLynx V4.0 (Micromass).



Quantitative analysis of the fractionated globin sample was performed by SRM. CID of precursor ions of m/z 833.9 and m/z 841.9, i.e., of the ions corresponding to single charged ions of Pyr-heptapeptide and Pyr-(2H8)heptapeptide, respectively, was carried out using argon as the collision gas at a pressure of 3.5 × 10-3 mbar and a collision energy of 48 eV. Product ions monitored and used for the quantification of the peptides were m/z 158.2 and 166.2 for Pyr-heptapeptide and Pyr-(2H8)heptapeptide, respectively, interpreted as pyrrolidinium ions of alkylated N-terminal valine and (2H8)valine, respectively (cf. Results) (23). The response in the SRM analysis was tested for the Pyr-heptapeptide standard at concentrations of 60 nM to 0.6 mM. THB-Val Adduct Analysis. 1. Isolation of Adducts by the N-Alkyl Edman Method. Hb adducts were analyzed by the N-alkyl Edman method (30) with modifications in the extraction of the formed PFPTH according to Pe´rez et al. (21). Twenty milligrams of globin sample from the experimental animals (diluted with 10 mg of a control globin to obtain a sufficient amount of globin for workup) was dissolved in formamide (1 mL). The internal standard dihydroxy(2H5)propyl-globin [(2H5)DHP-globin] (50 µL, 100 pmol), NaOH (40 µL, 1 M), and PFPITC (7 µL) were added, and the samples were left on a rocking mixer at room temperature overnight and then at 45 °C for 1.5 h. The formed THB-Val-PFPTH derivatives were extracted and acetylated according to Pe´rez et al. (21). Hydrophilic PFPTHs tend to be retained in the formamide and attached to glassware. Therefore, water was added to the formamide prior to extraction and the glassware used was silanized. To minimize losses of THB-Val-PFPTH, washing was reduced and extractions of the washing solution were repeated (21). The THB-Val-PFPTH was acetylated with acetic anhydride (25 µL), in acetonitrile (150 µL), in the presence of triethylamine (25 µL) for 15 min (31) (Figure 2). Before GC-MS analysis, the samples were dissolved in 50 µL of toluene. To establish calibration curves for the GC-MS/MS analysis of THB-Val adduct analysis, different amounts (0, 5.5-27.6 pmol and 0, 30-230 pmol) of a calibration standard, a heptapeptide with a known THB-Val adduct level (see Preparation of Peptide Standards), was added to samples of 30 mg of myoglobin and an equal amount of the internal standard (2H5)DHP-Val-globin (100 pmol). Derivatization and analysis of the samples for the two calibration curves were done as described above, at two different occasions. 2. GC-MS Analysis. GC-MS analyses were performed with a triple quadrupole MS (Finnigan TSQ700), coupled to a GC (Varian 3400) with a septum-equipped, temperature programmable injector (Varian SPI). The column used was a 30 m, 0.32 mm i.d., 1.0 µm thickness, fused silica capillary column (DB5MS; J&W Scientific), with a retention gap (2 m, medium polar column, 0.53 mm i.d.). The GC injector temperature program was as follows: 100-300 °C, 150 °C/min, and then isothermal at 300 °C for 18 min. Helium was used as the GC carrier gas. The temperature in the transfer line was 270 °C. The temperature program for the column was as follows: 1 min at 100 °C, 20 °C/min to 240 °C, 10 °C/min to 300 °C, and 300 °C for 7 min. The quantitative GC-MS/MS analyses were performed in the ECNI mode. Methane was used as the reagent gas. The pressure in the ion source was 5.5 Torr, and the temperature was 120 °C. The electron energy was 70 eV. The collision gas for MS/ MS was argon, and the pressure was 1 mTorr. Quantitative measurements were done by SRM of the acetylated THB-ValPFPTH and (2H5)DHP-Val-PFPTH using the product ions m/z 303 and m/z 342 formed from precursor ions m/z 534 and m/z 384, respectively (20, 21). The precursor ions m/z 534 and m/z 384 were formed after the loss of HF ([M - 20]-) and F + (CH3)2(CH)2CO from the valyl residue ([M - 103]-). The retention times were about 16.3 min for the analyte and 14.5 min for the internal standard.

Figure 4. CID spectrum of Pyr-heptapeptide standard (precursor ion m/z 833.9) corresponding to the globin R-chain in rat (cf. Table 1). A predominant formation of the pyrrolidinium ion m/z 158.2 is observed.

Results Standards and Hb Samples Prepared and Analyzed. The different standards and Hb samples prepared through in vitro and in vivo treatment are summarized in Table 1 together with adducts analyzed and methods for characterization/quantification of adduct levels. Characterization of Peptide Standards. The peptides (corresponding to rat and mouse N-terminal peptides) alkylated in vitro with DEB were characterized by LC-ESI-MS. Although the amino acid sequence varies between mouse and rat heptapeptides with regard to two amino acids, their molecular weights are the same. The m/z values of the molecular ions in the LC-ESI-MS analysis of the Pyr adduct in the two heptapeptides were therefore identical. The retention times in the HPLC analyses are, however, somewhat different. The DEB used was a racemic mixture [(2R,3R)- and (2S,3S)-DEB]. Two diastereoisomeric forms of Pyr- and THB-modified peptides were separated by HPLC in a similar ratio, as the valine component was a single enantiomer (S) (Figure 3). Full scan MS of the in vitro-alkylated heptapeptides revealed three singly charged peptides with m/z 851.9, 747.7, and 833.9 (Figure 3), corresponding to THBheptapeptide, unmodified heptapeptide, and Pyr-heptapeptide, respectively, eluting in this order (cf. ref 23 for mouse peptide). In MS/MS analysis of Pyr-heptapeptides (m/z 833.9), the predominating product ion (100%) was m/z 158.2, interpreted as the pyrrolidinium ion (1isobutylidene-3,4-dihydroxypyrrolidinium ion) formed in breakage of the bond between the isobutyl and the carbonyl of the modified N-terminal valine (Figure 4). The corresponding ion from the internal standards of Pyr(2H8)octapeptides was m/z 166.2 formed from CID of the molecular ions m/z 943 and m/z 929 from rat and mouse, respectively. SRM analysis of Pyr-heptapeptide at different concentrations within the range for quantification of samples shows a linear concentration-response curve. The limit of detection (LOD) for the Pyr-heptapeptide standard was about 300 fmol at the present analytical conditions. To determine the concentrations of Pyr- and THBmodified peptides in the peptide mixture by HPLC-UV according to Fred et al., the unmodified (2H8)octapeptide and heptapeptide were analyzed at different concentra-

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Fred et al.

Table 2. Comparison of Pyr-Val and THB-Val Adduct Levels after Treatment with DEB (cf. Figures 6 and 7) and EBdiol in Vivo and in Vitroa in vivo adduct level (nmol/g per mmol/kg) administered compound

adduct monitored

in vitro adduct level (nmol/g per mM h)

rat

mouse

rat

mouse

( 10 (n ) 7)

( 1.1 (n ) 3) c 105 ( 33 (n ) 3) 6.3 ( 2.9 (n ) 4) 24, 33

51, 59

47

0.30

ND

23

21

DEB

Pyr-Val

45d

DEB

THB-Val

EBdiol

THB-Val

23 ( 3.6 (n ) 6) 27, 30

58b

Values are means ( SD. No mean was calculated when only two values were available. The number of animals used is presented within parentheses. The measured Pyr-Val adduct level (cf. Figure 6 and ref 23) in the R-chain globin is multiplied by a factor 1.5, due to the relatively more reactive N-terminal valine in the β-chain (34), to give adduct levels per g globin comparable with levels of THB adduct; ND, not determined. b Mice treated with 0.150 mmol/kg body wt; data from Kautiainen et al. (23). c Mice treated with 0.300 mmol/kg body wt; data from Kautiainen et al. (23). d At the lowest dose, the adduct level could be quantified in only one of the three treated animals (cf. Figure 6). If the data point is excluded, the mean adduct level per administered dose does not change. a

Figure 6. Pyr-Val adduct levels (nmol/g R-chain globin) after i.p. injection of racDEB in rats. Three animals per administered dose were used for quantification. In two of the rats at the lowest treatment dose, the adduct level was below the LOD.

Figure 5. LC-MS/MS analysis of globin sample from DEBtreated rat (200 µmol/kg body wt). (A) Trace of the product ion at m/z 166.2 from the precursor at m/z 841.9 [internal standard; Pyr-(2H8) heptapeptide]. (B) Trace of the product ion at m/z 158.2 from the precursor at m/z 833.9 (Pyr-heptapeptide).

tions to establish a concentration-response curve for the respective peptide. It was assumed that the alkylated and unalkylated peptides have the same response in UV (220 nM). The degree of Pyr modification varied within a range of 30-40% in the different peptides. The degree of THB modification was about 20% in all in vitroalkylated peptides. Quantification of Hb Adducts after in Vitro and in Vivo Treatment with DEB. 1. Pyr-Val Adduct. Pyr-Val adduct levels were measured in globins from DEB-treated rats and from in vitro DEB treatment of rat and mouse erythrocyte hemolysates. The results from the analysis of globin samples from in vivo-treated mice were published earlier (23). Tryptic digests of globin were fractionated by HPLC-UV, and fractions consisting of Pyr-heptapeptide were pooled and analyzed by LC-MS/ MS (Figure 5). On the basis of the ratios between product ions corresponding to the pyrrolidinium ion of the Nterminal valine from Pyr-heptapeptide and from the

internal standard [Pyr-(2H8)heptapeptide], respectively, the adduct levels were quantified. The Pyr-Val adduct level in Hb from mouse hemolysate treated in vitro with DEB (150 mM h) was 47 nmol/g globin per mM h. The corresponding adduct levels in Hb from rat hemolysate treated in vitro (30 and 150 mM h) were 51 and 59 nmol/g globin per mM h, respectively (Table 2). Pyr-Val adduct levels after i.p. treatment with racDEB in rats are approximately the same per administered dose (45 nmol/g per mmol/kg body wt) as the one observed at the lower dose in mice (58 nmol/g per mmol/kg body wt) (Figure 6 and Table 2). A somewhat higher adduct level per administered dose is obtained in mice at the high dose (105 nmol/g per mmol/kg body wt). The LOD (s/n ratio ) 3) for monitoring of Pyr-Val adducts in rat Hb is approximately the same as in mouse Hb (∼1 nmol/g globin) (23). In blood from rats treated with the lowest dose of DEB (100 µmol/kg), the adduct levels were on the borderline for quantification of the PyrVal adduct; therefore, results from only one of the three rats treated with the lowest dose could be quantified (Figure 6). 2. THB Adducts. THB-Val adducts in rat and mouse Hb were quantified by GC-MS/MS analysis after detachment by the N-alkyl Edman method (Figure 2). The acetylated THB-Val-PFPTH elutes as a single peak in the GC-MS/MS analysis of samples from racDEB (R,R and S,S stereoisomers)-treated animals. In animals treated with EBdiol (R,R, S,S, R,S, and S,R stereoisomers), an additional peak elutes with an identical daughter fragmentation pattern. This peak, separating into a double peak, is also detected as a minor peak in



30 nmol/g per mmol/kg body wt) and rat (24 and 33 nmol/g per mmol/kg body wt).

Discussion

Figure 7. Calibration curve for THB-Val adduct levels analyzed by GC-MS/MS. Concentrations of in vitro-modified THBheptapeptide are plotted against the area peak ratio of the THBVal-PFPTH and the internal standard (IS) (2H5)DHP-ValPFPTH.

Figure 8. THB-Val adduct levels, after i.p. injection of racDEB, were about 3.3 times lower per administered dose in Hb from mice as compared to rats. This indicates a more effective hydrolysis of DEB in rat than in mouse; THB-Val is a measure of EBdiol, i.e., half-hydrolyzed DEB.

the globin from racDEB-treated animals, probably due to contamination of mesoDEB. These results are in agreement with Pe´rez et al. (21). Adduct levels were obtained from the ratio of the peak areas of the analyte (sum of the peaks) and the internal standard [(2H5)DHPVal-PFPTH] and the calibration curve. The calibration curve for adduct levels of acetylated THB-Val-PFPTH was linear (R2 ) 0.99, n ) 10) within the applied range of 0-8 nmol/g globin (Figure 7) and did not vary between different occasions for preparation and analysis. In rat globin from the in vitro DEB treatment, the THB-Val adduct level was 0.3 nmol/g globin per mM h. Because of similarities in reactivity between rat and mouse globin, the relative THB adduct level in mouse is expected to be approximately the same as in rat globin (experiment not conducted due to limited material). In the in vivo treatment of racDEB, the THB-Val adduct levels increased linearly with the administered dose and were about 3-4 times higher per administered dose in Hb from rats (23 nmol/g globin per mmol/kg body wt), as compared to mice (6.3 nmol/g globin per mmol/kg body wt) (Figure 8 and Table 2). This difference is significant (P < 0.001); see Discussion below. In the complementary study with in vitro and in vivo treatment with EBdiol, the THB-Val adduct levels were analyzed and compared to the results of the DEB treatment (Table 2). The THB-Val adduct levels after in vitro treatment with EBdiol were 23 and 21 nmol/g globin per mM h in rat and mouse globin, respectively. In the EBdiol-treated animals, the THB-Val adduct levels per administered dose were about the same in mouse (27 and

Viewpoints on Analytical Method. DEB is presumably the most potent carcinogenic metabolite in BDexposed animals. It forms a ring-closed adduct (Pyr-Val) in the reaction with N-terminal valine in Hb (20, 32). In preceding studies (23), a method based on LC-MS/MS of modified N-terminal peptides of the R-chain after tryptic degradation of globin was developed for measurement of the Pyr-Val adduct in DEB-exposed mice. The method is a complement to the extensively used N-alkyl Edman procedure, which is applicable for the measurement of monoepoxide metabolites of BD but not for the analysis of adducts such as Pyr-Val where the N terminus is blocked for the coupling/cleavage reaction with the reagent (PFPITC). The primary aim of the present study was to further develop the method for analysis of PyrVal adducts for application to rats exposed to racDEB and to compare the results with levels of THB adducts analyzed by GC-MS/MS according to the N-alkyl Edman method (21). The applicability of the method for analyzing the PyrVal adduct is facilitated by the adduct specific product ion formed from the ring-closed valine adduct in heptapeptides giving a high response in the MS/MS analysis. The predominant formation of the pyrrolidinium ion is favored by charge retention at the tertiary nitrogen (Figure 4), also observed in analysis of peptides containing the amino acid proline (33). A cyclic pyrrolidine adduct at an internal amino acid side chain (e.g., lysine) would neither give the same retention time nor result in a MS/MS fragment at m/z 158 (more likely m/z 116). In addition, the MS/MS analysis of the internal standard, a deuterium-substituted Pyr-(2H8)octapeptide [substituted with (2H8)valine at the N terminus], gives a pyrrolidinium ion 8 Da higher, i.e., the fragment formed is exclusively from a Pyr-modified valine. With the MS instrument used in this study, the best sensitivity was obtained with singly charged peptides, which were therefore chosen as the precursor ions in the MS/MS analysis. The relative abundance of singly and doubly charged peptides has been shown to be associated with the introduction of the sample to the mass spectrometer (direct or on-column), probably due to solvent effects, and also to vary between MS instruments. In the method development, problems such as poor reproducibility and degradation of the peptides were overcome by using plastic tubes and vials and by always storing samples in the freezer. However, the LOD in the analysis of Pyr-Val adducts in globin is still relatively high, which may be due to insufficient enrichment of the modified peptides. Problems with coeluting compounds that interfere in the MS/MS analysis were encountered in analysis using the doubly charged peptides. This is not due to coelution of the N-terminal peptides of the R- and β-chains, which have different retention times. The problem was even more pronounced in attempts at using the β-chain of Hb from mouse and rat (Fred, unpublished results), which led us to focus the present method development on R-chain adducts. Otherwise, the β-chain has the advantages that the analyte (an octapeptide) is identical in mice and rats and that the same standards can be used for the different species. Furthermore, the

792


levels of Pyr-Val adducts are higher (by about a factor 2) in the β-chain than in the R-chain (cf. Table 2 and ref 34). The mean adduct level per gram of globin is obtained by multiplying the measured adduct level in the R-chain by a factor of 1.5. The choice of internal standards and calibration standards presents difficult issues in Hb adduct measurement. Modified peptides to be used as standards can be synthesized to fit a specific analyte, characterized by LCMS, and the degree of modification can be determined using HPLC-UV, according to the procedure applied in this paper.2 In the measurement of adducts as modified peptides, it was found advantageous to use as an internal standard a peptide that also undergoes enzymatic degradation. Theoretically, there could be some difference in recovery between a heptapeptide originating from globin and the octapeptide constituting the internal standard, since a rat globin R-chain has to undergo up to 14 cleavages with trypsin, which cleaves at Arg and Lys, to give rise to the N-terminal heptapeptide. In the determination of THB adduct levels according to the N-alkyl Edman method, it was advantageous to use the characterized THB-modified heptapeptide as a calibration standard for the establishment of a calibration curve. THB adduct levels in DEB- and EBdiol-treated rats have previously been measured by Pe´rez et al. (21). As compared to that study, the observed adduct levels per administered dose are in the present study 2-3 times higher. This difference might, at least partly, be due to the use of different calibration standards. THB adducts could, in principle, have been analyzed by LC-MS/MS as tryptic heptapeptides. This was done for peptide standards and for in vitro samples (data not shown). However, an adduct specific product ion, facilitating analysis, is not formed from the THB adduct to the same extent as the pyrrolidinium ion from the Pyr adduct in the MS/MS analysis of modified heptapeptides; therefore, sufficient sensitivity was not obtained for analysis of the in vivo samples by this procedure. Several published papers deal with LC-MS analysis of modified peptides for characterization of alkylation patterns in proteins (34-37). There is, however, little work done on the quantitative measurement of Hb adducts in vivo based on levels of modified peptides and LC-MS. For a more general application of the method applied here, procedures for the enrichment of the modified peptides have to be improved. However, the method has been applied in a parallel study by Fred et al. (unpublished results)3 analyzing, even with improved LOD for in vivo samples, a methylpyrrolidine formed from isoprene diepoxide in mice treated with isoprene monoxide. Relevance of Observed Adducts. The terminal amino group is a major reactive site in globin chains of human Hb (18). The reaction rates for the formation of the measured adducts to N-terminal valines in Hb can be calculated from the data on adduct levels and doses from the in vitro reactions of Hb with DEB and EBdiol in Table 2 (18). Because Val-NH2 is primary, it retains a nucleophilic reactivity after the first reaction with the 2 Fred, C., Cantillana, T., Hendersson, A., Golding, B. T., and To¨rnqvist, M. Adducts to N-terminal valines in Hb from isoprene diepoxide, a metabolite of isoprene. Rapid Commun. Mass Spectrom. Submitted for publication. 3 Fred, C., Grawe ´ , J., and To¨rnqvist, M. Hb adducts and micronuclei in rodents after treatment with isoprene monoxide or butadiene monoxide. Mutat. Res. Submitted for publication.

Fred et al.

bifunctional DEB in favor of fast formation of a chemically stable pyrrolidine ring (20) (Figure 2). The reaction rates for Pyr-Val formation were determined to be 5.5 × 10-5 L g-1 h-1 for rat Hb and 4.7 × 10-5 L g-1 h-1 for mouse Hb, respectively. The corresponding reaction rates for the formation of the THB adduct from EBdiol are 2.3 × 10-5 and 2.1 × 10-5 L g-1 h-1 for rat Hb and mouse Hb, respectively. Ethylene oxide is a well-studied compound in this context and could be used for comparisons. For this compound, the reactivity toward N-terminal valine in rat Hb has been determined to be 5.8 × 10-5 L g-1 h-1 and for mouse Hb, it was determined to have a 12% lower rate (13). It would be expected that DEB has about the same reactivity as ethylene oxide. EBdiol, with one primary reactive epoxide carbon, is expected to have about half the reactivity of ethylene oxide. The determined reaction rates for DEB and EBdiol with Hb from rat and mouse are in reasonable agreement with expectations. In in vitro reactions, the THB adduct level in Hb is about 100 times higher per dose (mM h) from the incubation with EBdiol as compared to the incubation with DEB (Table 2). From the knowledge of hydrolysis of other aliphatic oxiranes, the estimated hydrolysis of DEB to EBdiol (t1/2 about 100 h, pH 7.4) during the in vitro incubation would be around 1% (38). This is in agreement with the observed relative THB adduct level in vitro after DEB incubation and calculated reaction rate (cf. above and Table 2). These results strengthen the earlier suggestion that Pyr-Val is a specific monitor of in vivo doses of DEB; THB-Val rather serves as a monitor of doses of EBdiol. Viewpoints on Metabolism. Although the primary aim of this study concerned the applicability of the Pyr adduct as a measure of dose of DEB in rat, the comparison with THB adduct levels may be used to elucidate differences between rats and mice in the elimination of DEB. The in vivo dose (obtained from adduct level and reaction rate) could be used for the calculation of elimination rates (18). DEB is rapidly distributed within the body, which justifies the application of a one compartment model in toxicokinetic considerations (17). From data in Table 2, the rate of elimination of DEB is calculated to be λ ) 1.2 h-1 (t1/2 ) 35 min) in the rat. A somewhat lower value was estimated in the mouse (λ ) 0.80 h-1, t1/2 ) 52 min, at low dose) from earlier measurements (23). At the highest dose in the DEB-treated mice, a saturation of the detoxication is indicated (not significant) as higher adduct levels (23). The THB adduct level per administered dose of DEB is significantly higher in rats than in mice (Figure 8). This was assessed by testing the differences in slopes in a linear regression model. The adduct levels for rats were down-scaled in order to adjust for the somewhat higher rate of formation in rats than in mice (cf. Table 2). The ratio between the slopes was estimated by nonlinear regression, and its 95% confidence interval was estimated by the profile likelihood method. The in vivo dose of EBdiol per administered dose of DEB was 3.3 times higher (95% CI: 2.3-5.8, P < 0.001) in rats than in mice. A comparison of Pyr and THB adduct levels after DEB treatment in vitro and in vivo suggests a faster enzymatic catalysis (by EH) of the hydrolysis of DEB to EBdiol in the rat as compared to the mouse, in agreement with


results of Boogaard et al. (39). However, the in vivo dose of DEB (inferred from Pyr-Val adduct levels) in mice and rats was approximately the same after treatment with DEB. In mice, a slower detoxication of DEB by EH catalysis is possibly compensated by a more effective conjugation to glutathione (40). The rate constants for elimination of EBdiol are calulated to be λ ) 0.80 h-1 (t1/2 ) 52 min) in rat and λ ) 0.74 h-1 (t1/2 ) 56 min) in mouse. For comparison, the in vivo half-life of ethylene oxide in rat (Lewis) is about 10 min at low doses administered i.p. (41). A comparison of THB-Val adducts in mice and rats after exposure to DEB (by inhalation) was performed earlier by Swenberg et al. (22). Considering the different experimental conditions, the results are in agreement with the present study. They observed a relatively higher THB-Val adduct level (by about a factor of 2) in the rat as compared to the mouse with an increasing treatment dose of DEB. [It should be noted that no adjustment of the adduct levels due to the duration of exposure and the life span of the erythrocytes was performed (22).] The opposite relation is observed in the same study where THB adduct levels are measured in mice and rats after treatment with BD (up to 62.5 ppm for 2 weeks). The relatively higher THB-Val adduct level in mice gets even higher (by about a factor of 8) at a higher exposure level (1000 ppm BD for 13 weeks) (42). The measurement of blood concentrations of EB and DEB in comparison with THB adduct levels indicates that in BD exposure and, particularly in rats, EBdiol is rather formed by epoxidation of butenediol than by hydrolysis of DEB (cf. Figure 1) (1). For a reliable cancer risk estimation of BD, the dose of DEB per exposure dose of BD in humans needs to be estimated. The measurement of Pyr-Val adducts in Hb would offer a useful tool. However, further development toward higher sensitivity of the analytical method is required. At present, THB adducts may serve as a surrogate monitor of BD exposure, using an established Pyr-Val/THB-Val adduct level ratio (and its variation) for preliminary estimation of in vivo doses of DEB.

Acknowledgment. We acknowledge Prof. Em. Lars Ehrenberg for critical reading of the manuscript, Dr. Fredrik Granath for statistical analysis, Dr. Per Rydberg for synthesis work, and Anna-Lena Magnusson for skillful technical assistance. We thank the Department of Analytical Chemistry at Stockholm University for giving us access to the LC-MS instrument and B.Sc. Helena Idborg for support in the analysis. The study was supported financially by grants from the Swedish Energy Agency, the Swedish Research Council for Environmental Agricultural Science and Spatial Planning, The Swedish National Board for Laboratory Animals, and the Magnus Bergwall Foundation.

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