Association of Dehydromonocrotaline with Rat Red Blood Cells

The association of radiolabeled monocrotaline pyrrole (DHM) with red blood cell (RBCs) ghosts ... Extensive washing of the RBCs with isotonic phosphat...
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Chem. Res. Toxicol. 1997, 10, 694-701

Association of Dehydromonocrotaline with Rat Red Blood Cells Michael W. Lame´,† A. Daniel Jones,‡ Dexter Morin,† Dennis W. Wilson,§ and H. J. Segall*,† Departments of Veterinary Molecular Biosciences and of Veterinary Pathology, Microbiology and Immunology and Facility for Advanced Instrumentation, University of California-Davis, Davis, California 95616 Received October 9, 1996X

The association of radiolabeled monocrotaline pyrrole (DHM) with red blood cell (RBCs) ghosts, globins, and heme was examined to determine their role in the transport and stabilization of this hepatic produced putative toxic metabolite of the pyrrolizidine alkaloid monocrotaline (MCT). Rats were administered 5 mg of DHM/kg, iv, and RBCs and plasma were harvested at 4 and 24 h. Extensive washing of the RBCs with isotonic phosphate buffer did not decrease the amount of radioactivity associated with the cells. The level of DHM equivalents recovered in the RBCs did not decrease between 4 and 24 h, while the plasma levels, which were 29- and 75-fold lower, respectively, decreased from 5.0 to 2.2 nmol of DHM equiv/g of plasma. Globin chains were found to contain 383 and 453 pmol of DHM equiv/mg of protein, respectively. Rats receiving 10 mg of DHM/kg, iv, with RBCs collected at 2 h, had approximately double the level of radioactivity associated with their RBCs in addition to 2 times the amount of adducts on the globin chains. Globins and ghosts plus heme (2 h) contained 69% and 2% of the radioactivity, respectively. Globin chains treated with an acidic ethanol solution containing AgNO3 resulted in the removal of 31% of the associated radioactivity. GC/ MS and TLC separation of AgNO3-displaced material revealed the presence of the ethyl ether derivatives of 7-hydroxy-1-(hydroxymethyl)-6,7-dihydro-5H-pyrrolizine. The HPLC separation of globin chains revealed that the majority of radioactivity coeluted with the β-chains. In conclusion, this study found that the administration of radiolabeled DHM resulted in extensive radioactive labeling of RBCs; similar findings have been reported for [14C]MCT.

Introduction Monocrotaline (MCT) is a pyrrolizidine alkaloid (PA) produced by the plants Crotalaria spectabilis and Crotalaria retusa (1). A single injection of MCT can produce a pulmonary vascular syndrome in rats that is characterized by pulmonary hypertension, cor pulmonale, and proliferative pulmonary vasculitis (2-5). The pulmonary effects induced by MCT make it a useful model for studying human primary pulmonary hypertension. MCT, as well as other PAs, requires metabolic activation in hepatic tissue to exert toxic effects. Activation requires the dehydrogenation of MCT to the putative toxic metabolite dehydromonocrotaline (DHM), or monocrotaline pyrrole (6). Reindel et al. (7) and Pan et al. (5) have shown that synthesized DHM (1.0-3.5 mg/kg) could produce a delayed (14 day) and progressive microvascular leakage, interstitial inflammation, and alterations in the blood vessel smooth muscle that resulted in pulmonary hypertension. The pulmonary changes observed by these investigators are similar to the alterations noted following the administration of the parent alkaloid, MCT. These studies support the idea that DHM is the toxic hepatic metabolite of MCT transported to pulmonary sites. However, due to the exceedingly short half-life of DHM in aqueous media, the pyrrole must be intrave* Author to whom correspondence should be addressed. Fax: 916752-4698. E-mail: [email protected]. † Department of Veterinary Molecular Biosciences. ‡ Facility for Advanced Instrumentation. § Department of Veterinary Pathology, Microbiology and Immunology. X Abstract published in Advance ACS Abstracts, May 15, 1997.

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nously administered in an aprotic solvent to produce pulmonary insult (8). The unstable nature of DHM in aqueous media raises the question of whether the liver could produce and transfer sufficient pyrrole to the blood stream to overcome rapid hydrolysis of DHM to monocrotalic acid and the slow-reacting alkylating agent dehydroretronecine. Prior distribution studies conducted with [14C]MCT demonstrated that red blood cells (RBCs) could potentially provide a medium for the transport of reactive electrophiles produced in the liver to remote organs. Estep et al. (9) observed that RBCs contained high levels of 14C following either intravenous or subcutaneous administration of [14C]MCT. RBCs collected 4 h postsubcutaneous injection contained more than 10-fold as much radioactivity as plasma. The t1/2 for the β-phase of radioactivity was 6-fold higher for the RBCs relative to the plasma. Pan et al. (10) showed that RBCs previously circulated (90 min) through an isolated perfused liver in buffer containing [14C]MCT, subsequently washed, and recirculated through an isolated lung preparation could transfer electrophiles to pulmonary tissue. The present report will, for the first time, examine the distribution of necine base tritium-labeled and uniformly labeled [14C]DHM following intravenous administration. Special attention will be given to DHM’s relationship with the various components of the RBCs, ghosts, heme, and globin chains. These studies will aid in determining if RBCs could act as a carrier of DHM produced at hepatic locations for transport to pulmonary sites. © 1997 American Chemical Society

Interaction of Dehydromonocrotaline with RBCs Chart 1. Structures of Monocrotaline (MCT), Dehydromonocrotaline (DHM), and Ethyl Ethers of 7-Hydroxy-1-(hydroxymethyl)-6,7-dihydro5H-pyrrolizine (DHP)a

a MCT is a macrocyclic diester composed of the amino alcohol retronecine and monocrotalic acid.

Methods Caution: The toxicological effects of dehydromonocrotaline (DHM) are not fully understood; however, the reagent is a powerful alkylating agent and should be handled carefully to minimize exposure. Chemicals. All chemicals and supplies unless otherwise noted were obtained from Fisher Scientific (Santa Clara, CA). Chart 1 contains structures referred to in the text. Uniformly labeled monocrotaline ([14C]MCT) (0.4 mCi/mmol) was obtained from C. spectabilis plants grown under a confined atmosphere of 14CO2; 14CO2 was produced by the addition of NaH14CO3 (American Radiolabeled Chemicals Inc., St. Louis, MO) to 18 N H2SO4 (11). [3H]MCT (0.357 mCi/mmol) was also obtained from C. spectabilis by administrating [2,3-3H]putrescine (Du Pont, Wilimington, DE) using the wick method (12). The location of the tritium in MCT was determined by Ba(OH)2 hydrolysis according to the method of Adams et al. (13). Retronecine (uncorrected melting point, 117-118 °C) derived from this procedure was purified by the methods of Hoskins et al. (14). Radioactivity was found to be restricted to the necine base of MCT as previously reported (15). Radiolabeled MCT was extracted as previously reported (11, 16); purity (g99%) was determined by HPLC using a Hamilton PRP-1 column (150 × 4.1 mm, 5 µm; Hamilton, Reno, NV). Conditions were 5% CH3CN:95% NH4OH (10 mM), isocratic for 15 min followed by a linear gradient to 25% CH3CN:75% NH4OH (10 mM); flow rate 1 mL/min. One minute fractions were collected for scintillation counting. Radiolabeled dehydromonocrotaline (DHM) was prepared according to the methods of Mattocks et al. (17) by dehydrogenation of either [3H]- or [14C]MCT with o-bromanil (tetrabromo-1,2-benzoquinone) (Aldrich, Milwaukee, WI). The resulting product was recrystallized from a mixture of hexane and anhydrous ethyl ether. The ethyl ether derivatives of 7-hydroxy-1-(hydroxymethyl)-6,7-dihydro-5H-pyrrolizine (DHP) were synthesized according to the method of Culvenor et al. (18), but instead of using dehydrosenecionine, DHM was substituted as previously described by Mattocks and Jukes (19). Briefly, 26.25 mg of DHM dissolved in 250 µL of N,N-dimethylformamide (DMF, anhydrous; Aldrich) was added to a stirring solution of ethanol. The reaction was allowed to progress at room temperature for 5 h, then 1 mL of 10% K2CO3 was added, the solvents were removed under reduced pressure, and 10 mL of H2O was added. The aqueous mixture was extracted with ethyl ether and the ether layer dried over Na2SO4 and K2CO3. The reaction yielded a mixture of 7-ethoxy-1-(ethoxymethyl)-6,7dihydro-5H-pyrrolizine and 7-ethoxy-1-(hydroxymethyl)-6,7-dihydro-5H-pyrrolizine. The pyrrole adduct of Gly-His-Lys was synthesized by mixing 2.6 µmol of the peptide acetate salt (Sigma, St. Louis, MO) in 1 mL of water with 2.6 µmol of DHM delivered in 20 µL of DMF.

Chem. Res. Toxicol., Vol. 10, No. 6, 1997 695 The peptide solution was adjusted to pH 7 with NH4OH prior to the addition of DHM. The reaction was allowed to progress for 1 h at 4 °C; prior to analysis samples were stored at -80 °C. Tissue Distribution. Male Sprague-Dawley rats (Charles River, Wilmington, MA), mean weight 154 g (n ) 4), received an intravenous bolus of [14C]DHM (5 mg/kg, 5 mg/500 µL of DMF) and were housed in silanized glass metabolism cages (Bio-Serv, Frenchtown, NJ); they were allowed food and water ad libitum. Animals were euthanized 24 h after injection with an overdose of pentobarbital, and the tissues were harvested. Organs were homogenized in 1 volume of distilled H2O; an aliquot was dissolved in Soluene 350, Hionic Fluor scintillation cocktail (Packard, Meriden, CT) was added, and samples were allowed to stand at room temperature for 24 h prior to being assayed for radioactivity by scintillation counting. Red blood cells were subjected to oxidation prior to scintillation counting using an Ox-300 tissue oxidizer (R.J. Harvey Instrument Corp., Hillsdale, NJ). Covalent binding involved the solubilization of tissue homogenates in 2% sodium dodecyl sulfate (SDS) at 70 °C for 1 h followed by exhaustive dialysis of tissue samples retained in Spectrapor-3 dialysis bags (3500 MW cutoff) against 0.1% SDS, 10 mM sodium phosphate buffer (pH 7) (20). Following SDS dialysis, samples were further dialyzed against two changes of distilled water and then dissolved in 1 N NaOH prior to scintillation counting. Protein concentrations were determined according to the methods of Lowry et al. (21). An additional single animal received the same treatment, but its tissues were harvested at 72 h. Distribution of [3H]DHM Equivalents in RBCs, Ghosts, Globins, and Heme. Three groups of animals were used in this study. Two groups (n ) 5) of animals received a single iv bolus of [3H]DHM (5 mg/kg, 69.1 mg/mL of DMF) and were sacrificed at 4 and 24 h. Mean body weights and standard deviations were 247 ( 8 and 225 ( 11 g, respectively. The third group of animals (175 ( 8 g, n ) 4) received 10 mg of [3H]DHM/ kg and were sacrificed 2 h after injection. These latter animals were injected once per day, and both the heme and ghosts of the red blood cells were prepared the same day; for the 4 and 24 h experiments only the globin chains were recovered from the RBCs. Blood in all experiments was collected under pentobarbital anesthesia from the abdominal vena cava into syringes containing ACD solution (7.3 g of citric acid, 22 g of sodium citrate, and 24.5 g of dextrose, adjust volume to 1 L). RBCs were removed from whole blood by centrifugation at 1000g for 20 min; plasma was stored (-20 °C) for later analysis. RBCs were washed with 3 × 10 vol of 310 mOsm sodium phosphate buffer (pH 7.4). Approximately 1 g of RBCs was resuspended in 1 mL of isotonic phosphate buffer; RBCs were then hemolyzed with a stream of 20 mOsm phosphate buffer (pH 7.4). The rupture of cells was carried out at 4 °C with a 1:25 (w/w) ratio of cells to hypotonic solution, disregarding the amount of isotonic buffer used to resuspend the cells. Ghosts were removed from the solution by centrifugation (5900g, 40 min, 4 °C). Ghosts were washed with 2 × 25 mL of 20 mOsm phosphate buffer followed by 25 mL of distilled water. Ghosts were then frozen at -80 °C prior to the removal of excess liquid by lyophilization. The procedure for the preparation of ghosts was a minor modification of that reported by Dodge et al. (22). To prepare heme free globins, the supernatants from the hemolysis and the first wash of the ghosts were slowly added to a stirring solution of 400 mL of 5 mM HCl in acetone (-20 °C). The globin chains from approximately 1 g of RBCs were washed with 3 × 50 mL of acetone (-20 °C). For the 4 and 24 h studies, the globin chains were initially dried under a stream of N2 and then further dried under reduced pressure. Globin chains from the 2 h study, following repeated washings with acetone, were resuspended in H2O and dialyzed (3.5 kDa MW cutoff) against 0.1% NaHCO3 (2 × 3 L) followed by 50 mM sodium phosphate buffer (pH 7.4) (2 × 3 L). This was followed by additional dialysis against 2 × 3 L of distilled water. Contents of the bag were lyophilized to dryness. The hemecontaining acetone solution was filtered through Whatman #1

696 Chem. Res. Toxicol., Vol. 10, No. 6, 1997 Scheme 1. Mechanism for Removal of Protein Thiol-Linked DHP with Acidic AgNO3 Solution and Subsequent Formation of Ethyl Ethers of DHPa

Lame´ et al. Table 1. Tissue Distribution and Covalent Binding (24 and 72 h) of [14C]DHM (5 mg/kg, iv) in Sprague-Dawley Rats covalent bindingc

distribution tissue plasma RBC liver lung kidney heart muscle

24

ha

1.5 ( 0.1 95.1 ( 5.3 4.1 ( 0.4 23.7 ( 2.4 11.6 ( 0.7 10.2 ( 1.1 1.0 ( 0.2

72

hb

0.5 68.5 2.8 5.2 5.5 5.5 NA

24 h

72 h

37 ( 6 700 ( 83 40 ( 3 365 ( 33 126 ( 6 125 ( 14 13 ( 4

28 589 35 118 70 88 NA

a Tissue distribution expressed as nmol of [14C]DHM equiv/g ( SD (n ) 4). b Represents a single animal. c Covalent binding expressed as pmol of [14C]DHP equival/mg of protein ( SD (n ) 4). a

Adapted from Mattocks and Jukes (19).

filter paper, and the filtrate was reduced to dryness under reduced pressure. The residue was transferred with acetone to Teflon tubes and reduced to dryness under a stream of N2. The heme was washed with 3 × 5 mL of distilled water before lyophilizing again. Globin chains were analyzed for radioactivity either directly or following oxidation. Both ghosts and heme samples were oxidized prior to assaying for the presence of 3H. Analysis of AgNO3 Displaceable Globin Chain Adducts. Globin chains from the 2, 4, and 24 h experiments utilizing [3H]DHM and an additional animal treated with 20 mg of [14C]DHM/ kg (blood harvested 2 h after injection) were used in these studies. Globin chains (40-110 mg) were resuspended in 20 mL of absolute ethanol and sedimented by centrifugation. Globin chains were again uniformly resuspended in 20 mL of ethanol, and 500 mg of AgNO3/1 mL of water was added to the stirring solution; this was immediately followed by 1 mL of 5% trifluoroacetic acid (19). The silver salt in this step was used to remove thioether adducts formed in the reaction of DHM with globin chains. Scheme 1 depicts the mechanism for this displacement. The reaction was allowed to progress for 1 h at room temperature before the addition of 2 mL of 25% K2CO3 followed by an additional 10 min of stirring. The solution was centrifuged and the supernatant removed from the pellet. The pellet was washed twice with 20 mL aliquots of ethanol; washes were pooled with the original supernatant. The ethanol was then filtered through silanized glass wool. Prior to scintillation counting aliquots of ethanol were added to Safety-Solve counting cocktail (Research Products International Corp., Mount Prospect, IL). The ethanol insoluble precipitate was oxidized prior to scintillation counting. For structural determinations, the ethanol extracts were reduced to dryness in silane-treated pear-shaped flasks under reduced pressure. The residue was transferred from the flasks with 4 × 2 mL of H2O to the Teflon tube. The flask was then washed with 3 × 10 mL of ethyl ether which were successively used to extract the aqueous layer. The ether layer was dried over Na2SO4 and Na2CO3, and aliquots were reduced to dryness and redissolved in ethyl acetate. Aliquots of this and reference standards were separated by TLC on silica gel plates (Redi/Plt Sil, silica gel G) using 50% heptane:50% acetone as a solvent system. The presence of pyrroles was revealed by spraying the plates with Ehrlich reagent that was composed of 2 mL of boron trifluoride etherate plus 2 g of 4-(dimethylamino)benzaldehyde (Aldrich) dissolved in 100 mL of ethanol. Plates were scanned in reflectance mode (565 nm) with a Shimadzu densitometer (Columbia, MD). An additional sample, separate from that examined by TLC, was analyzed by GC/MS (EI+) using a VG Trio-2 mass spectrometer (VG Masslab, Altrincham, U.K.), ionization energy 70 eV. Separations were performed on a DB-1 column (30 m × 0.25 mm i.d., film thickness 0.25 µm; J&W Scientific, Folsom, CA), flow rate 1 mL of helium/min, injector 250 °C, column initial temperature 100 °C (1 min hold) followed by a ramp (10 °C/ min) to 250 °C (5 min hold). Separation and Identification of [14C]DHM-Exposed Globin Chains. Globin chains, from an animal receiving 20

mg of [14C]DHM/kg, iv, were separated on a Vydac C18 column (5 µm, 10 × 250 mm, 300 Å pore; Western Analytical, Temecula, CA); conditions: flow rate 5 mL/min, isocratic (5 min) 35% CH3CN:65% H2O:0.1% TFA followed by a 35 min linear gradient to 55% CH3CN:45% H2O:0.1% TFA. Fractions (1 min) were collected for scintillation counting, and additional fractions were collected for electrospray ionization mass spectrometry (ESI/ MS). Fractions to be used for MS were lyophilized and stored at -80 °C. Prior to analysis samples were resuspended in 50% H2O:50% CH3CN (1 mg of protein/mL). Samples (10 µL) were analyzed on a VG/Fisons Quattro-BQ triple quadrupole mass spectrometer (VG Biotech, Altrincham, U.K.). Samples were delivered directly using an Isco µLC-500 syringe pump (Isco, Lincoln, NE) set at 10 µL/min, solvent 50% CH3CN:50% H2O with 0.1% formic acid. Spectra were collected in positive ion mode with a capillary voltage of 3.5 kV, source temperature 65 °C, and cone voltage at approximately 35 V. Analysis of Gly-His-Lys Pyrrole Adducts and the Nonadducted Peptide. Sample solutions (2 µL) mixed with 2 µL aliquots of 50% dithiothreitol:50% dithioerythritol or 3-nitrobenzyl alcohol (Aldrich, Milwaukee, WI) were analyzed using a ZABHS-2F mass spectrometer (VG Analytical, Manchester, U.K.). Fast atom bombardment (FAB) ionization was carried out using a beam of xenon atoms (8 keV, 1 mA). Collisionally activated dissociation/mass analyzed ion kinetic energy experiments were performed to obtain structural information. The first sector of the instrument was tuned to the mass of the [M + H]+ or the [M - H2O + H]+ ion, and the helium collision gas (1 × 10-6 mbar) was introduced into the field-free regions of the instrument for collisional activation. The scanning of the electric sector produced a spectrum of the fragment (product) ions.

Results Tissue Distribution and Covalent Binding. Data for the 24 and 72 h studies are reported in Table 1. The highest levels of radioactivity were associated with the RBCs for both time periods; RBCs also had the highest levels of covalent adducts. The plasma compartment contained very low levels of 14C, and the levels of covalent binding were equally low. For the 24 h period, 82 ( 5% and 3 ( 1% of the administered 14C were excreted in the urine and feces, respectively. The 72 h animal excreted 88% of the administered radioactivity in the urine with 80% being excreted within the first 24 h. Distribution of [3H]DHM Equivalents in RBCs, Ghosts, Globins, and Heme. Extensive washing of RBCs with isotonic sodium phosphate did not lower the amount of [3H]DHM equivalents associated with these cells. Both the 4 and 24 h animals had essentially the same level of tritium associated with their RBCs. However, the level of tritium in the plasma compartment was seen to drop significantly with respect to time; between the 4 and 24 h intervals this compartment decreased by approximately 2.3-fold. A doubling of the dose in con-

Interaction of Dehydromonocrotaline with RBCs

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Table 2. Distribution of [3H]DHM Equivalents in Blood time (h)a

RBCb

RBC (washed)c

plasmad

globine

% AgNO3 displacedf

2 4 24

288 ( 15 155 ( 15 157 ( 14

328 ( 21 146 ( 16 164 ( 13

18.9 ( 1.9 5.0 ( 0.7 2.2 ( 0.2

870 ( 86 383 ( 38 453 ( 43

30.7 ( 3.2 27.6 ( 1.6 34.8 ( 1.6

a Time after intravenous administration of [3H]DHM and the collection of blood. All animals received 5 mg of [3H]DHM/kg except the 2 h group (10 mg of [3H]DHM/kg). n ) 4 for the 2 h group; n ) 5 for the 4 and 24 h time points. b RBCs separated from plasma by centrifugation and directly decomposed in a tissue oxidizer. Data expressed in nmol of [3H]DHM equiv/g. c After removal of plasma, RBCs washed with 3 × 10 volumes of isotonic sodium phosphate buffer. Data expressed in nmol of [3H]DHM equiv/g. d Data expressed as nmol/g; corrected for dilution with ACD solution. The 4 and 24 h means are significantly different using a two-tailed Student’s t-test (p < 0.0001). e Globins prepared as described in Methods. Binding expressed as pmol of [3H]DHM/ mg of protein. Identical number of samples as indicated under (a) except only four data points for the 4 h animals. f Percent of total recovered radioactivity displaced from globin chains by AgNO3; see Methods.

junction with a decreased time (2 h) resulted in an approximate doubling of the [3H]DHM equivalents associated with RBCs. The amount of [3H]DHM equivalents covalently linked to the globin chains for the 4 and 24 h animals was essentially the same, while the increase in dose for the 2 h animals resulted in a 2-fold increase in adducts associated with globin chains (Table 2). The separation of the 2 h RBCs into ghosts, globin chains, and heme revealed the following. The ghosts had 236 ( 26 and the heme 226 ( 47 pmol of [3H]DHM equiv/mg (gravimetric measurement) associated with them (n ) 4). With respect to washed RBCs, globin chains represented 68.5 ( 8.7% and heme + ghosts 1.6 ( 0.1% of the radioactivity. With respect to the 4 h (n ) 4) and 24 h (n ) 5) studies, the globin chain fraction was found to contain 58.0 ( 4.1% and 67.2 ( 1.9%, respectively, of the original radioactivity in the RBCs. Globin Chain Adducts Displaced with AgNO3. Results from the 2, 4, and 24 h animals are recorded in Table 2. The percentage of radioactivity displaced by AgNO3 was essentially the same for all time points; the mean for combined time points was 31%. For the identification of the adduct displaced by silver, a single rat administered 20 mg of [14C]DHM/kg, with globins harvested 2 h after injection, was used. Globin chains in this study had 2.58 nmol of [14C]DHP equiv/mg of protein associated with them. For 41.5 ( 1.2 mg of protein (n ) 3) treated with AgNO3, 34.0 ( 5.4% of the radioactivity attached to the globin chains was recovered in the ethanol extract. Following removal of the ethanol under reduced pressure, resuspension in water, and extraction with ethyl ether, 91.7 ( 0.7% of the 14C present in the ethanol was recovered. Total recovered 14C (adducts removed by AgNO3 plus nonremovable) was 87.5 ( 13.6% of the original radioactivity present on the protein. For the silica gel separation of AgNO3-displaced adducts, 107 mg of protein was used. The final residue remaining after removal of the ether was yellow in color. A sample of 15 nmol of [14C]DHP equiv/3.5 µL of ethyl acetate was spotted on silica gel plates along with the standard. Development with Ehrlich reagent revealed the presence of two spots in the standard at Rf 0.69 and 0.57 for 7-ethoxy-1-(ethoxymethyl)-6,7-dihydro-5H-pyrrolizine (I) and 7-ethoxy-1-(hydroxymethyl)-6,7-dihydro5H-pyrrolizine (II), respectively. The sample contained

Figure 1. TLC (silica gel) separation of di- (I) and mono- (II) ethyl ether derivatives of DHP: (A) standards and (B) AgNO3displaced material from globin chains. Axis (x) denotes vertical travel of the stage in the densitometer. TLC plate was positioned so that larger x-axis values denote areas closer to the origin.

Figure 2. GC/MS (EI+) total ion chromatogram of synthetic standards of I and II, retention times 10.21 min (scan 610) and 8.27 min (scan 494), respectively. See Chart 1 for structures.

one Ehrlich positive spot with an Rf value of 0.68, corresponding to I (Figure 1). The appearance of a single spot was indicative of the entire conversion of globinreleased pyrrole to the diethyl ether derivative of DHP. Mattocks and Jukes (19) have previously found that under acidic conditions the bulk of the product formed in this reaction was the diethyl ether derivative; in the absence of acid the reaction primarily yields the 7-ethoxy derivative. We have noted a substantial degree of variability for this reaction since a separate sample, subjected to the same conditions of derivatization, analyzed by GC/MS contained a substantial quantity of the 7-ethoxy product. For analysis by GC/MS, 109 mg of globins was extracted; the resulting residue was resuspended in ethyl acetate to give 12.7 µg of [14C]DHP equiv/100 µL of solvent. The standard’s total ion chromatogram (Figure 2) showed two major peaks at 8.27 and 10.21 min. The present sample contained two peaks with identical retention times to those seen for the standard. The sample

698 Chem. Res. Toxicol., Vol. 10, No. 6, 1997

Figure 3. Electron impact mass spectra of II - H2O obtained from (A) a synthetic standard and (B) AgNO3-displaced material derived from globin chains.

Lame´ et al.

Figure 4. Electron impact mass spectra of I obtained from (A) a synthetic standard and (B) AgNO3-displaced material derived from globin chains.

Chart 2. Potential Structures of Ions Observed in Figures 3 and 4

Figure 5. HPLC separation of globin chains obtained from an animal administered 20 mg of [14C]DHM/kg, iv. Peaks 2-5 were found to have masses of 15 848, 15 845, 15 847, and 15 902 Da, respectively; peak 1 contained two components with masses 15 143 and 15 187 Da. Dashed line represents the radioactive profile.

peak (8.27 min) had an m/z 163 ion corresponding to the loss of water from II (Figure 3). The peak at 10.21 min showed an m/z 209 corresponding to the molecular ion of I (Figure 4). Both spectra contained m/z 117, 118, 119, and 120 common ions seen in the fragmentation of pyrroles formed by the dehydrogenation of the necine base portion of pyrrolizidine alkaloids. Chart 2 shows the potential structures of the ions appearing in Figures 3 and 4. Structures for m/z 117, 118, 119, 120, and 134 ions have previously been advanced (23). Separation of Globin Chains by HPLC. ESI/MS of the peaks obtained from the HPLC separation of the bulk globins (Figure 5) revealed that peak 1 was com-

posed of two components having masses 15 143 and 15 187 Da. Peaks 2-5 had masses 15 848, 15 845, 15 847, and 15 902 Da, respectively. The first peak had a mass that corresponded to the R-chain, while peaks 2-5 had masses similar to those previously reported for variants in the β-chain (24). The radioprofile (Figure 5) indicated that the bulk of the radioactivity corresponded to the region containing the β-chains. In this study we were unable to distinguish the increase in mass of either the R- or β-chain that would be suggestive of alkylation by a pyrrole. Globin chains used in this study had 2.58 nmol of [14C]DHP equiv/mg of protein; a ratio of 2.58 nmol of adduct/64 nmol of globin (mass ∼15 600 Da) or approximately 4% of the protein would be alkylated. Gly-His-Lys-DHP and Nonadducted Peptide Spectra. Results of the FAB MS/MS experiments (Fig-

Interaction of Dehydromonocrotaline with RBCs

Figure 6. FAB MS/MS product spectrum of (A) dehydrated Gly-His-Lys-DHP and (B) Gly-His-Lys. Structures depict the positions of fragmentation leading to the corresponding ions in the spectra. The position of the pyrrole on the imidazolium group of histidine is considered the most likely site of adduction but cannot be inferred from the spectrum.

ure 6A) revealed the presence of a dehydrated monoadduct of Gly-His-Lys-DHP; the [M + H - H2O]+ had an m/z value of 458. The product ion spectrum did not reveal the site of adduction of DHP containing only the ion at m/z 118 previously seen in DHP adducts of glutathione and cysteinylglycine (25, 26) and the ion at m/z 341 corresponding to the peptide Gly-His-Lys. In the absence of the adduct the peptide was seen to fragment extensively (Figure 6B).

Discussion Estep et al. (9) reported that the administration of [14C]MCT (60 mg/kg, sc) resulted in the extensive labeling of RBCs relative to other tissues, 85 and 49 nmol equiv of MCT/g, 4 and 24 h after injection, respectively, while plasma levels were significantly less, 8 and 2 nmol/g, respectively. The intravenous administration of [14C]MCT indicated that the radiolabel associated with RBCs had a β-phase half-life of 13.4 h compared to 2.2 h for plasma. Results of the present study also showed the dramatic labeling of the RBCs relative to the plasma. However, the administration of 5 mg of [14C]DHM/kg, iv, produced approximately 2 times the level of radiolabel in the RBCs obtainable with 60 mg of [14C]MCT/kg, sc, after 24 h. The 24 h level of covalent binding to the RBCs for [14C]DHM was 2.8 times the level obtained with [14C]MCT 4 h postinjection (9). A comparison between RBCs extensively washed with isotonic phosphate and unwashed cells from the 2, 4, and 24 h animals showed that washing was unable to reduce the level of radiolabel associated with these cells. Previous studies have shown that RBCs were capable of

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augmenting the covalent binding in pulmonary tissue. Tandem liver-lung perfusions in the presence of MCT, with and without RBCs, indicated that 182 and 97 pmol of MCT equiv/mg of lung protein were covalently bound, respectively, for a 1 h experiment. These studies implicated RBCs as a potential medium for the transport and stabilization of electrophilic metabolite(s) from the liver to pulmonary sites (10). Our inability to detect a component associated with RBCs capable of being exchanged with other tissues could be because the pyrrole stabilized by RBCs was removed prior to the time points examined. It is also possible that DHM is stabilized by the lipophilic membranes of the RBCs and removal in the pulmonary system requires a close association with other lipophilic structures such as the cellular membranes of the endothelial cells. At a covalent binding level of 182 pmol of MCT equiv/mg of protein, with a 1.3 g lung, and assuming 18% protein, 43 nmol of adduct/ lung could be expected. To attain this level of binding in the tandem liver-lung perfusion, containing 100 mL of medium and 40 µmol of MCT, only 0.1% of the dose as DHM would ideally need to be transported by RBCs. The level of pulmonary covalent binding for in vivo studies was approximately 3 times less (9) than reported for the tandem isolated perfused liver-lung. Both the in vitro and in vivo studies with MCT indicated that the amount of electrophile necessary to produce pulmonary damage was probably low in comparison to the administered dose. This is additionally supported by studies (5) showing that a single dose of 1 mg/kg DHM could result in pulmonary hypertension after 21 days. Provided the exchange of electrophiles between the RBCs and pulmonary sites (covalent interactions) is an efficient process and pulmonary insult is related to adduct formation, the amount of exchangeable material carried by RBCs would be minor in comparison to the standard dose of MCT needed to precipitate pulmonary hypertension. The detection of these potentially low levels of transportable and exchangeable electrophiles would definitely be difficult. We are presently exploring the transportation and stability capabilities of RBC membranes by preparing ghosts of RBCs and comparing their ability to produce pulmonary insults when administered in vivo with and without DHM and then comparing these values to the administration of DHM in plasma. With respect to RBC distribution (2 h), approximately 2% and 69% of the original tritium was recovered in the heme plus ghosts and globin chains, respectively. Both heme and ghosts shared an equal percentage of the radiolabel. The total recovered tritium in this study is probably not a reflection of what is potentially transportable and exchangeable but losses incurred during the lysis of the RBCs and repeated washing of the ghosts, heme, and globins followed by extensive dialysis of the globins which ultimately resulted in the loss of the components of the RBCs and their associated adducts. The 24 h distribution study with [14C]DHM also indicated that dissolution with SDS followed by extensive dialysis did not displace radioactivity from the components of the RBCs (absence of detectable 14C outside the dialysis membrane). This treatment indicated that at least by 24 h it is unlikely that an exchangeable metabolite(s) was associated with the RBCs. Treatment of globins with AgNO3 showed that at least 31% of the adducts were formed through the interaction of the pyrrole with sulfhydryl groups on the globin chain(s). Mattocks and Jukes (19) have previously shown that

700 Chem. Res. Toxicol., Vol. 10, No. 6, 1997

an acidic, ethanolic solution of AgNO3, mechanism depicted in Scheme 1, was capable of removing pyrroles from the elements of whole blood with the subsequent formation of ethyl ether derivatives of DHP. These pyrrolic derivatives were analyzed as in the present report by TLC and GC/MS. Mattocks and Jukes (19) have used the AgNO3 technique for detecting the presence of sulfur-bound adducts in whole blood (4 mL) spiked with DHM (10 mg) as well as adducts recovered from rats receiving a 1 week prior ip injection of MCT (5 mg/kg). This technique, however, only revealed the potential contribution of protein sulfhydryl-adduct interactions; the use of radiolabel in the present report indicates that 70% of the adducts formed could be with other nucleophiles. Using the model peptide, Gly-His-Lys, we were able to show that reactions with DHM produced a monoadduct. Location of the site of adduction was not evident from product ion spectra that were composed of only two ions corresponding to the original peptide and m/z 118 corresponding to the pyrrole. The modification of His or Lys residues by DHM would explain our inability to recover all the bound pyrrole by AgNO3 treatment of globin chains. Mattocks and Bird (27) have indirectly shown that dehydroretronecine (DHR, 7βhydroxy-1-(hydroxymethyl)-6,7-dihydro-5H-pyrrolizine) could react with adenine and guanine derivatives, histidine, and tryptophan. Tomer et al. (28) and Wickramanayake et al. (29), using fast atom bombardment (FAB) and tandem mass spectrometry, have shown that DHR could react with the N6, O2, and N2 positions of adenosine, thymidine and thymidine 5′-monophosphate, and deoxyguanosine 5′-monophosphate, respectively. Under in vitro conditions DHR has been shown to crosslink plasmid and viral DNA (30). HPLC separation of the globin chains revealed that the bulk of the radioactivity was recoverable from the region containing the β-chains. Our inability to detect these adducts, which are estimated to occur in 4% of globin chains, by ESI/MS is due to a combination of factors. First, the radioactive profile indicated that the bulk of the adducts was within the region containing the four β-chains; adducts could be spread over these four forms. DHM is also a bifunctional alkylating agent capable of forming interchain cross-links. This property would also be expected to result in a decrease of sample homogeneity. The third factor making detection difficult is that increases in mass can either come as m/z 135 or 117 depending on whether the pyrrole moiety dehydrates (25, 26). An additional factor, which is evident from the examination of the daughter ion spectrum of Gly-HisLys-DHP, is the absence of extensive fragmentation. This spectrum contained only the m/z 118 and 341 ions. Product ion spectrum of the original peptide, however, shows extensive fragmentation at a number of sites. The spectrum is suggestive of the ease at which the pyrrole adduct is removed from the peptide. This facile loss may indicate the potential instability of pyrrole adducts in the charged and gaseous states leading to premature dissociation. A combination of these factors would make detection by ESI/MS and the associated deconvolution software difficult at best. Previous investigators have also reported the selective binding of electrophilic metabolites of xenobiotics to both the human and rodent β-chain of hemoglobin (31-35). β-Cysteine-93, a site that is conserved in mammalian hemoglobin, was found to be a common site of adduction (33, 36). β-Cysteine-125 in rat hemoglobin (31) and β-histidine-143 and R-histidine-

Lame´ et al.

20 from human hemoglobin (36) have also been shown to be modified by xenobiotics. Naylor et al. (37) found that approximately 80% of the adducts formed between benzo[a]pyrenediol epoxide and human hemoglobin were through ester linkages with unidentified carboxylate group(s). Such adducts were stable to the extent of being able to survive extensive ethyl acetate and butanol extraction followed by 10 days of PBS dialysis at 4 °C. Following hydrolysis by trypsin, adducts were recovered as benzopyrenetetrols with the remaining radioactivity (15%) being associated with peptides. However, adducts formed to mouse globins were much more labile and did not survive extensive cleanup procedures. Pyrrole adducts (at least 71%) in the present study survived the cleanup procedure. Pan et al. (10) have shown that RBCs previously circulated (90 min) through hepatic tissue, in the presence of MCT, were capable of transporting and exchanging an unknown electrophile(s) at pulmonary sites. This transport and formation of covalent adducts occurred even after the RBCs had been extensively washed before their recirculation through pulmonary tissue. These adducts could have originated from the modification of the large pool of covalently linked pyrrole associated with the globin chains. Mattocks and Jukes (19) have previously hypothesized that pyrroles could react with protein methionine residues producing transportable but labile sulfonium salts that could exchange pyrrole for other nucleophilic sites. Additional support for the concept that adducted proteins could act as a medium for the transport and exchange of electrophilic metabolites comes from preliminary studies in this laboratory. These studies have focused on the in vitro formation of pyrrole adducts with glutathione transferase(s). Reactions with a mixture of glutathione transferases and radiolabeled DHM revealed, upon HPLC separation, the close association of radiolabel with the UV absorbance profile of the individual glutathione transferases. When individual isozymes were reacted with DHM and subsequently digested with trypsin, a different result was obtained. HPLC separation of the resulting peptides produced a number of sharply resolved UV-absorbing components. However, the radioprofile did not correspond to the UV profile that was composed of a number of radiolabeled peaks superimposed on a large background of radioactivity. Results of this study indicated that some of the peptide adducts, when removed from the environment of the intact protein, were not stable. Whether this instability is an isolated phenomenon related to the interaction of the peptide adduct and the large surface area of the Vydac C18 column under acidic conditions is presently unknown. Examination of the spectra shown in Figure 6 indicates that in the absence of the pyrrole adduct extensive fragmentation is seen to occur, producing major ions at m/z 110, 167, 195, and 212; with the addition of the pyrrole to the peptide this fragmentation is limited to the production of ions at m/z 118 and 341. This facile loss of the pyrrole, highly favored over the fragmentation of the peptide, is suggestive of the vulnerability of the pyrrole bond. If this physical instability observed in the charged and gaseous states is applicable to biological systems, the pyrrole adducts may be particularly susceptible to removal from globin chains following either enzymatic degradation or environmental changes observed during circulation through hepatic and pulmonary tissues. The potential for hemoglobin, other proteins, or degradation products of these proteins acting

Interaction of Dehydromonocrotaline with RBCs

as reservoirs for the transport and exchange of pyrroles warrants further investigation.

Acknowledgment. This work was supported by NIH Grant HL48411. We are grateful to Sheila Lame´ for the typing and editing of the manuscript.

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