Chem. Res. Toxicol. 1992,5, 386-391
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Utilization of Glutathione during 1,2-Dihaloethane Metabolism in Rat Hepatocytes Paul A. Jean and Donald J. Reed* Department of Biochemistry and Biophysics, Oregon S t a t e University, Corvallis, Oregon 97331-6503 Received October 28,1991
The metabolism of 1,2-dihaloethanes (DHEs) to glutathione-containing metabolites by freshly isolated rat hepatocytes was investigated. 1,2-Dichloroethane (DCE), 1,2-dibromoethane (DBE), and 1-bromo-2-chloroethane (BCE) were metabolized to S-(2-hydroxyethyl)glutathione(HEG), S-(carboxymethy1)glutathione (CMG), and S,S'-(1,2-ethanediyl)bis(glutathione)(GEG). The formation of these glutathione-containing metabolites was concomitant with the depletion of intracellular glutathione (GSH) and accounted for 58%, 84%, and 71% of the DCE-, BCE-, and DBE-induced loss of intracellular GSH, respectively. The covalent binding of [14C]DBE to hepatocyte protein reached 18.7 nmol/mL of cell suspension (7.8 nmol/mg of protein) within 2.0 h of incubation. Half of this covalent binding occurred within 0.5 h of incubation (4.0 nmol/mg of protein) in the presence of high levels of intracellular GSH (30% of initial GSH level a t 0.5 h). Hepatocyte metabolism of 2-chloroacetic acid produced only CMG. 2-Chloroethanol metabolism gave rise to CMG and HEG in a 11.51.0 ratio; 2-chloroacetaldehyde produced almost equal amounts of CMG and HEG. GEG formation was increased significantly for DBE and BCE when GSH was added to the medium during treatment, suggesting that the GSH conjugates S-(2-haloethyl)glutathioneare exported from the hepatocytes. These results indicate that the glutathione S-transferase-catalyzed conjugation of GSH with the DHEs is responsible for the majority of the DHE-induced GSH depletion. The S-(2-haloethyl)glutathioneconjugates appear responsible for the extensive covalent binding to protein observed during [ 14C]DBE metabolism.
I ntroductlon The l,2-dihaloethanes (DHEs)' have become an important class of compounds due to their unique chemical and physical properties. 1,2-Dibromoethane(DBE) and l,%-dichloroethane(DCE) have proved useful as industrial solvents, raw material for vinyl halide synthesis, fumigants, and gasoline additives (1,2).However, such varied and extensive utilizations have raised concern for their potential adverse effects on humans and the environment. DBE and DCE are suspected human carcinogens on the basis of their demonstrated mutagenicity in a variety of test systems (3,4)and carcinogenicity in laboratory animals (5,6). They have also been shown to be toxic to laboratory animals, and accidental human exposures have been lethal (7-9). Mixed-function oxidation (MFO)and direct conjugation with GSH are recognized as the two principle pathways for DHE metabolism. The MFO metabolism produces chemically reactive 2-haloacetaldehydesthat may bind to cellular macromolecules or undergo further metabolism to the respective 2-haloethanol or 2-haloacetic acid (IO, 11). The direct conjugation of DHEs to GSH, as catalyzed by the glutathione S-transferases (GSTs), yields a sulfur half-mustard, S-(2-haloethyl)glutathione (11, 12). Recently, in vivo formation of S-(2-chloroethyl)glutathione was observed in the bile of BCE-treated rats (13).Sulfur mustards are well-known as potent alkylating agents, and the ability of S-(2-chloroethyl)glutathioneto alkylate a variety of functional groups common to protein or DNA has been demonstrated (14). GSH, the predominate nonprotein thiol in most cells, is well-known for its participation in the detoxification of activated oxygen intermediates and electrophilic metabolites of xenobiotics (15,16).However, in DHE metabolism,
* To whom correspondence should be addressed.
GSH may function in both detoxification and bioactivation. The alkylation of GSH (instead of cellular macromolecules) by reactive DHE metabolites would constitute a detoxification process whereas the formation of glutathione sulfur half-mustards represents bioactivation. The participation of GSH in either of these processes as related to the mediation of DHE toxicity is not well-defined. We report herein on the use of freshly isolated hepatocytes to investigate the utilization of GSH in DHE metabolism. DHE-induced GSH depletion was characterized and found to be concomitant with the formation of at least three glutathionecontaining DHEderived metabolites and extensive protein covalent binding. The relationship between GSH depletion and specific pathways for DHE metabolism is discussed.
Materials and Methods Chemicals. Glutathiooe (GSH), y-glutamylglutamate, glutathione disulfide (GSSG), and 1,2-dibrom0[1,2-'~C]ethane(13.3 mCi/mmol) were purchased from Sigma Chemical Co. (St. Louis, MO). DCE, BCE, iodoacetamide, 2-chloroethanol, 2-chloroacetic acid, and 2-chloroacetaldehyde were purchased from Aldrich Chemical Co. (Milwaukee, WI). DBE was purchased from Eastman Kodak Co. (Rochester, NY). Caution: 1,Z-Dihalo-
ethanes have been shown to be carcinogenic in laboratory animals and may be carcinogenic in humans. Carcinogenic and radioactive substances should be handled with due caution. Isolation of Hepatocytes. Hepatocytes were isolated from male Sprague-Dawley rata (180-210 g) as described by Fariss et al. (17). Hepatocytes prepared in this way were better than 90% viable as assessed by trypan blue exclusion.
Lactate de-
l Abbreviations: GSH, reduced glutathione;DCE, 1,2-dichloroethane; BCE, 1-bromo-2-chloroethane;DBE, 1,2-dibromoethane; HEG, S42hydroxyethy1)glutathione; CMG, S-(carboxymethy1)glutathione;GEG, S,S'-(1,2-ethanediyl)bis(glutathione); PCA, perchloric acid; DHE, 1,2dihaloethane;GST, glutathione S-transferase;MFO, mixed-function oxidation; HPLC, high-performanceliquid chromatography;FAB, fast atom bombardment.
Q893-228~/92/27Q5-Q386$Q3.QQ/Q 0 1992 American Chemical Society
Glutathione Metabolites of 1,2-Dihaloethanes hydrogenase leakage was assessed as described (17)with a Kontron UVicon 810 spectrophotometer. Incubation of Hepatocytes with DHEs and Chlorinated Derivatives. Freshly isolated hepatocytes were resuspended to a concentration of 2.0 X lo6 cells/mL in a modified Fischer’s medium that lacked all sulfur amino acids except for methionine (0.67 mM) and included 3.5 mM calcium, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid, and 20-60 mM y-glutamylglutamate as an internal standard for HPLC analysis. No significant loss of y-glutamylglutamatewas observed throughout the cell incubations. Ten milliliters of cell suspension was placed into 25mL Erlenmeyer flasks, and an aliquot of agent was added. The DHEs were added directly to the flasks whereas the chlorinated derivatives were prepared in water just prior to addition. The flasks were sealed with rubber septums and incubated a t 37 O C for 1h in an orbital shaker. After incubation, aliquots of the cell suspension were taken and prepared for HPLC analysis. Other studies with the radiolabeled DBE were conducted as described above for the nonradiolabeled DHEs. The eluent from the HPLC ultraviolet detector was collect in 1-min fractions and mixed with Formula 969 scintillation cocktail (NEN Research Products, Boston, MA). The radioactivity was measured with a Package Tri-Carb liquid scintillation spectrometer. The quenching of samples required that each vial be counted before and after spiking with [14C]tolueneto determine counting efficiency. The covalent binding of [14C]DBEto hepatocyte protein was determined following incubation of cells with [14C]DBE (0.335 mCi/mmol) as above. At specific times 0.2 mL of 70% trichloroacetic acid was added to 1.2 mL of cell suspension followed by centrifugation for 1min in an Eppendorf centrifuge (No. 5415, Brinkmann) a t 14000 rpm. The supernatant was used for metabolite determinations as above. The protein pellet was washed with 1.0-mL aliquots of methanol until no more radioactivity could be detected in the wash. The washed protein pellets were then dissolved in 1.0 M sodium hydroxide. An aliquot of solubilized protein (0.3 mL) was added to the scintillation cocktail and the radioactivity determined. Protein estimations were performed with BCA protein assay reagent (Pierce, Rockford, IL)with serum albumin as standard. HPLC Analysis. To quantitate the formation of glutathione-containingmetabolites, 0.6 mL of cell suspension was added to 0.1 mL of 70% perchloric acid (PCA) in 1.5mL microfuge tubes. After vortexing and centrifugation (13000g for 1min) an aliquot of the supernatant was derivatized with Sanger’s reagent as described (18)except that iodoacetamide was subsituted for iodoacetic acid. Determination of intracellular GSH was performed as described by Fariss and Reed (18)with the noted exception. Briefly, 0.6 mL of cell suspension was placed over 0.4 mL of dibutyl phthalate that was layered over 0.5 mL of 10% PCA containing y-glutamylglutamate. Centrifugation for 1min a t 13000g was used to pellet “viable” cells through the oil layer and into the PCA. The amount of y-glutamylglutamate carried through the oil by the cells during centrifugation was found to be insignificant. Aliquots of the PCA supernatant were assayed as above. HPLC employed columns packed with an amine bonded phase prepared by derivatization of Spherisorb with (3-aminopropy1)silane; a binary mobile phase system consisting of 80% methanol and 0.8 M acetate in 80% methanol was used as described (14). Note that longer columns (4.6 mm X 300 mm) and a variety of gradient programs were used to aid in the resolution of components. Standards of the compounds of interest were derivatized as above for the cell samples and their retention times characterized with each column/gradient system just prior to analysis of cell samples. A Spectra-PhysicsHPLC pump and SP8700XR pump controller, Micrometrics autosampler Model 728, Spectroflow 757 UV detector, and Hewlett Packard integrator Model 3390A were used. Chemical Synthesis. The synthesis of HEG was carried out by adding excess 1-bromoethanol to GSH under alkaline conditions. Isolation of HEG from the reaction mixture was performed by preparative HPLC using a reverse-phase C18 column (Whatman ODSII, 20 mm X 500 mm) and 0.1% acetic acid as the mobile phase. HEG was collected upon elution from the column, frozen, lyophilized, and characterized by fast atom
Chem. Res. Toxicol., VoZ.5, No. 3, 1992 387
Control
DCE
BCE
DBE
Figure 1. DHE-induced depletion of glutathione in freshly isolated hepatocytes. Hepatocytes (2.0 X lo6 cells/mL) were treated with 1.2 mM DHE for 1h, and the intracellular GSH level remaining after incubation was determined. Values represent the mean of at least five experiments & SE. (*) Significantly different than the control 0,< 0.01). DCE = l,Zdichloroethane, BCE = l-bromo-2-chloroethane, DBE = 1,2-dibromoethane.
bombardment (FAB) mass spectrometry. Purity was judged to be better than 97% by HPLC, with the major contaminant being GSH. S,S’-(l,2-Ethanediyl)bis(glutathione)(GEG) was formed by mixing 2 equiv of GSH with 1equiv of DBE in 30% ethanol in the presence of triethanolamine, pH 9.0, at room temperature for 24 h (19). HPLC isolation and FAB mass spectrometry characterization of GEG from the reaction mixtures were performed as above with the exception of using 10% methanol and 0.1% acetic acid as the mobile phase. S-(Carboxymethy1)glutathione (CMG) was isolated from the reaction mixture in which 100 mg of GSH was added to 120 mg of iodoacetic acid in 5.0 mL of 0.5 M Tris base a t pH 9.1. Preparative HPLC was used to purify the CMG and FAB mass spectrometry for characterization, as was done for HEG. GSH reacts readily with 2-chloroacetaldehyde to form S(formylmethy1)glutathione (20). However, in that study, the isolation of S-(formylmethy1)glutathionefrom its reaction mixture was unsuccessful (20). Our attempts to isolate S-(formylmethy1)glutathione from reaction mixtures were also u n s u c c e s s ~
Results Depletion of Intracellular GSH. Exposure of freshly isolated hepatocytes to the various DHEs resulted in significant depletion of intracellular GSH by 1h as shown in Figure 1. DBE depleted GSH to 17% of control whereas BCE and DCE depleted GSH to 38% and 81% of control, respectively. The lack of lactate dehydrogenase leakage from the hepatocytes during this 1-h incubation indicates that DHE-induced depletion of GSH occurred without cell lysis. GSH Metabolites. Exposure of freshly isolated hepatocytes to DHEs gave rise to the formation of metabolites with retention times characteristic of HEG, CMG, and GEG. Figure 2 depicts the elution profile of 365 nm absorbing material and the elution profile of radioactivity for hepatocytes treated with radiolabeled DBE. The elution of HEG, CMG, and GEG standards under identical conditions is indicated. The quantity of each metabolite formed varied with the DHE as shown in Figure 3. Large
Res. Toxicol., VoZ.5, No. 3,1992
Jean and Reed
1.00 Q
e
Table I. Effect of Extracellular Glutathione (1.0 mM) on the Formation of S.S'-( 1.2-Ethanediy1)bis(pl1utathione)" agent GEG (% of control f SE) 1,2-dichloroethane 120.5 f 22.4 1-bromo-2-chloroethane 179.3bf 14.1 1,2-dibromoethane 161.4bf 12.3
-
0.75
-
P cn
a 0.50 Q
CI
"Freshly isolated hepatocytes were treated with 1.2 mM 1,2-dihaloethanes in the presence or absence (control) of 1.0 mM glutathione, and the quantities of glutathione-containing metabolites were determined. The values represent the effective change in metabolite formation by the inclusion of extracellular glutathione. The values represent the mean f SE for at least three separate experiments. GEG = S,S'-(1,2-ethanediyl)bis(glutathione). The amount of metabolite formed with the addition of extracellular glutathione was significantly different (p < 0.05) than control.
0.25 -
Le
-
V
'
0
lb
2'0
3b
4'0
5b
6b
7'0
Fraction
Figure 2. HPLC analyais of l,%dibromo[ 1,2-14C]ethane-treated hepatocytes. The upper chromatogram represents the elution of 365 nm absorbing material, and the lower chromatogram, radioactivity. Freshly isolated hepatocytes were treated and assayed as described in the Materials and Methods section. Peak assignments are based upon the elution times of standards. HEG = S-(2-hydroxyethyl)glutathione,CMG = S-(carboxymethyl)glutathione, GEG = S,S'-(1,2-ethanediyl)bis(glutathione).
* 20 -
T*
HEG
CMG
GEG
Metabolite
Figure 3. Quantitation of glutathione-containing metabolites produced by hepatocytes during a 1-h incubation with 1.2 mM l,2-dihaloethanes. Values represent the mean of at least five separate experiments f SE. (*) Significantly different from DCE (p < 0.01) and (**) significantly different than DCE and BCE (p < 0.05). (Dark-shaded bars) 1,2-dichloroethane; (hatched bars) 1-bromo-2-chloroethane; (light-shaded bars) 1,2-dibromoethane. HEG = S-(2-hydroxyethyl)glutathione, CMG = S-(carboxymethyl)glutathione, GEG = S,S'-( 1,2-ethanediyl)bis(glutathione).
amounts of HEG were produced following DBE and BCE treatment. The amount of HEG formed by DBE was not significantly different from BCE, whereas they were both significantlygreater than that produced by DCE. All three of the DHEs produced relatively small quantities of CMG. GEG formation followed a structure-activity relationship in that DBE yielded 1.5 times more GEG than BCE, which
Table 11. Metabolism of 1,t-DichloroethaneDerivatives to Glutathione-Containing Metabolites" nmol/mL of cell suspension (ISE) agent HEG CMG GEG ND 49.0 f 6.9 ND 2-chloroacetic acid ND 2-chloroethanol 3.1 f 1.0 35.8 f 3.0 ND 2-chloroacetaldehyde 23.0 f 2.6 29.2 f 2.8
" Freshly isolated hepatocytes were treated with 2-chloroethanol (0.5 mM), 2-chloroacetic acid (0.5 mM), and 2-chloroacetaldehyde (0.25 mM) for 1 h, and the amount of glutathione-containing metabolites were determined. Values represent the mean f SE for at least three separate experiments. ND = formation of metabolite was not detected. HEG = S-(Zhydroxyethyl)glutathione,CMG = S-(carboxymethyl)glutathione, GEG = S,S'-(1,2-ethanediyl)bis(glutathione).
yielded approximately 5 times more GEG than DCE. Effect of Extracellular GSH. Hepatocytes were exposed to the DHEs in the presence and absence of extracellular GSH to investigate the potential export of GSH sulfur half-mustards into the medium. Incubation of hepatocytes with 1.2 mM DCE, BCE, and DBE in the presence of 1.0 mM extracellular GSH increased the amount of GEG formation by- 120%, 179%, and 161% of control, respectively (Table I). The effect of extracellular GSH on HEG formation was less dramatic and varied with the DHE. CMG formation was not significantly different from that for controls. Metabolism of Derivatives of DCE to HEG, CMG, and GEG. The formation of glutathione-containing metabolites from 2-chloroethanol, 2-chloroacetic acid, and 2-chloroacetaldehyde is summarized in Table 11. GEG was not detected when hepatocytes were exposed to 2chloroacetaldehyde, 2-chloroacetic acid, or 2-chloroethanol. 2-Chloroacetaldehyde produces similim amounts of HEG (44%) and CMG (56%),whereas 2-chloroacetic acid yields only CMG. 2-Chloroethanol gave rise to predominately CMG (93%) with only a small amount of HEG (7%). Accountability of GSH Loss. We have shown in Figures 1-3 that exposure of freshly isolated hepatocytes to DHEs resulted in a loss of intracellular GSH and formation of at least three GSH-derived metabolites. Figure 4 represents a quantification of the total amount of glutathione found as GSH and oxidized glutathione (GSSG) as well as that for each of the three GSH-derived metabolites. The amount of GSH associated with metabolite formation accounts for 55% or more of the depleted GSH. However, the amount of glutathione present in these five forms could account for essentially all of the DCE- and BCE-induced GSH depletion. Covalent Binding of [14C]DBE to Protein. DBE metabolism caused extensive GSH depletion and covalent, binding to hepatocyte proteins (Table 111). The rate of covalent binding of [14C]DBEto hepatocyte protein de-
Chem. Res. Toxicol., VoZ.5, No. 3, 1992 389
Glutathione Metabolites of 1,2-DihaZoethanes
Table 111. Time-Dependent Accumulation of Glutathione-ContainingMetabolites and Covalent Binding during [14C]-l,2-Dibromoethane Metabolism by Isolated Hepatocytes" treated [nmol of GSH equiv/mL of cell suspension (ISE)] protein control covalent GEG bindingb time (min) GSH CMG GSH HEG 0 45.2 (f8.1) 45.2 (f8.1) 0.0 0.0 0.0 0.0 10.6 (f3.9) 5.3 (f0.6) 15 47.3 (17.6) 3.5 (f3.5) 4.9 (f0.7) 30.6 (f3.2) 14.6 (f3.7) 9.6 (f0.7) 30 52.8 (f8.8) 15.2 (f3.5) 9.2 (f1.4) 6.2 (f0.9) 16.4 (35.9) 14.4 (f0.8) 60 65.5 (f9.8) 14.1 (f2.2) 8.3 (f1.6) 8.1 (f1.2) 120 104.1 (f20.2) 28.4 (12.9) 11.1(f7.4) 21.2 (f7.0) 18.7 (f1.8) 3.9 (f3.9)
total 45.2 54.9 54.8 61.3 83.3
"These data represent at least three separate experiments. GSH = glutathione, HEG = S-(2-hydroxyethyl)glutathione,CMG = Scarboxymethyl-glutathione,GEG = S,S'-( 1,2-ethanediyl)bis(glutathione). Nanomoles of covalently bound 1,2-dibromoethane per milliliter of cell suspension based upon an estimated 2.4 mg of protein/mL of cell suspension (2.0 X lo6 cells/mL).
120
x
100 -
* 80 -
(3.9)
60 40 -
20 0-
-r
C
DCE BCE 1.2 mM 1,2-Dihaloethane
DBE
Figure 4. Quantity of glutathione recovered as glutathione (boldface hatches), glutathione disulfide (dark shading), S-(2hydroxyethy1)glutathione (lightface closely spaced hatches), S(carboxymethy1)glutathione(lightface widely spaced hatches), and S,S'-(l,Zethanediyl)bis(glutathione) (light shading) following l-h incubation with 1.2 mM 1,2-dihaloethane. Data are expressed as the percent of glutathione equivalences for control (no 1,2dihaloethane exposure). Values represent the mean ( M E ) of a t least three separate experiments. (*) Significantly different than control (p < 0.05). C = control, DCE = 1,2-dichloroethane,BCE = l-bromo-2-chloroethane, DBE = 1.2-dibromoethane.
creased with time of incubation and correlated with GSH depletion. After 2.0 h of incubation, a total of 18.7 nmol of [14C]DBEwas covalently bound per milliliter of cell suspension. Half of this total occurred within the first 30 min of incubation (9.6 nmol/mL of cell suspension). Intracellular GSH remained relatively high during this same time frame in that depletion after 30 min of incubation decreased the GSH level to 30% of the initial level. By 60 min of incubation, 77% of the total covalent binding that had accumulated was accompanied by depletion of intracellular GSH to 17% of initial levels. The second hour of incubation gave rise to almost complete depletion of GSH with [WIDBE covalent binding increasing from 14.4 to 18.7 nmol/mL of cell suspension.
Discussion Metabolism of the DHEs, DCE, BCE, and DBE, by freshly isolated hepatocytes resulted in DHE-induced depletion of intracellular GSH with the concomitant formation of at least three glutathionecontaining metabolites, HEG, CMG, and GEG. The depletion of GSH in hepatocytes is characteristic of the DHE-induced depletion of liver GSH in laboratory animals (21). Evidence in support
of the direct participation of GSH in in vivo DHE metabolism is suggested by the identification of DHE-derived mercapturic acids and other thiol-containing metabolites in the urine of DHE-treated animals (22,23) as well as the presence of S-(2-chloroethyl)glutathione in the bile of BCE-treated rats (13). In addition, HEG and GEG were detected in the liver and kidney tissues of DBE-treated rats (19). DHE metabolism occurs principally through two different pathways, both of which produce reactive intermediates. Figure 5 depicts the general outline of these metabolic pathways and the potential utilization of GSH in each. The formation of 2-haloacetaldehyde has been demonstrated for both DCE and DBE ( 1 0 , 2 4 ) . 2-Haloacetaldehydes can undergo rapid conjugation with GSH, as well as undergo further oxidation or reduction to the respective 2-haloacetic acid or 2-haloethan01(20). However, the conjugation of 2-haloethanol and 2-haloacetic acid with GSH is markedly slower than that for the 2-haloacetaldehydes and does not appear to be catalyzed by the GSTs (20). The S-(formylmethy1)glutathione that would be produced from the conjugation of GSH with 2-haloacetaldehyde may be oxidized to CMG or undergo reduction to HEG as suggested from the studies of 2chloroethanol metabolism (20). 2-Chloroethanol has been shown to be metabolized by purified enzyme preparations and subcellular fractions to CMG (20). The author proposed that CMG formation was the result of 2-chloroethanol oxidation to 2-chloroacetaldehyde and conjugation of 2-chloroacetaldehyde with glutathione, followed by oxidation of the S-(formylmethy1)glutathione to CMG. In our investigation, freshly isolated rat hepatocytes metabolized 2-chloroethanol to CMG and HEG. The amount of CMG formation was 11.5 times greater than that for HEG. However, 2-chloroacetaldehyde metabolism gave rise to almost equal amounts of CMG and HEG, suggesting that S-(formylmethyl)glutathione, the conjugation product of 2-chloroacetaldehyde with glutathione, readily undergoes both oxidation and reduction. The amount of CMG and HEG produced during 2-chloroethanol metabolism would be indicative of 2-chloroethanol metabolism to 2-chloroacetic acid prior to glutathione conjugation and not to 2chloroacetaldehyde. These observations are suggestive of a metabolic sequence different from. that proposed by Johnson (20). DBE and BCE metabolism gave rise to levels of HEG that were severalfold greater than CMG, whereas DCE produced equimolar amounts of HEG and CMG. These results suggest, on the basis of the metabolism of 2chloroacetaldehyde to HEG and CMG, that 2-haloacetaldehyde formation from DBE and BCE does not contribute substantially to the observed GSH depletion. 2Haloacetaldehyde formation would be expected to give rise
390 Chem. Res. Toxicol., Vol. 5, No. 3, 1992
Jean and Reed
OH
Figure 5. Proposed pathways of 1,2-dihaloethanemetabolism by hepatocytes. MFO = mixed-function oxidation, GSH = glutathione, HEG = S-(2-hydroxyethyl)glutathione,CMG = S-(carboxymethyl)glutathione,GEG = S,S'-(1,2-ethanediyl)bie(glutathione).
to equal amounts of HEG and CMG. The majority of HEG derived from BCE and DBE is most likely due to the GST-catalyzed formation and subsequent hydrolysis of the respective glutathione sulfur half-mustards. The apparent lack of 2-haloacetaldehyde formation (i.e., the dissimilarity in HEG versus CMG formation) from BCE and DBE suggests that the observed covalent binding of [14C]DBE to cellular protein was due to S- (bromoethy1)glutathione formation rather than 2-bromoacetaldehyde. DCE and DBE have been shown to be substrates for the GSTs (21,251. Studies of the isozyme-specificmetabolism of DBE by rat liver, kidney, and human liver forms have demonstrated that all of the forms tested exhibit some level of activity with DBE (25). However, the a-class isozymes were the most active for both rat and human. Metabolism of DHEs by GST catalysis could contribute substantially to the tissue-specific depletion of GSH, as indicated in Figure 5. The formation of GEG would be especially important in this regard. In our investigation, GEG formation accounted for a significant amount of the total DHE-induced depletion of GSH. GEG formation is thought to represent the alkylation of GSH by the respective glutathione sulfur halfmustards. The quantity of GEG produced during BCE and DBE metabolism would suggest that the hepatocytes are exposed to a substantial amount of glutathione sulfur half-mustard. Glutathione sulfur half-mustard formation is likely to be even greater than that indicated by the formation of GEG, considering that glutathione sulfur half-mustards rapidly hydrolyze to HEG as well as react with cellular constituents other than GSH. The half-life of S-(2-bromoethyl)glutathionewas determined to be less than 10 s (26)whereas that for S-(2-chloroethyl)glutathione was reported to be approximately 20 min (27). It would appear from the quantities of HEG and CMG produced from DBE and BCE that at least 80% of the HEG arises from the hydrolysis of the respective glutathione sulfur half-mustards, with the remainder resulting from the possible reduction of S-(formylmethy1)glutathione. Hepatocytes are well-known to export GSH and glutathione S-conjugates (28,291. The mutagenicity of bile from DCE-treated mice and perfused livers has been attributed
to the excretion of S-(2-haloethyl)glutathionefollowing its formation in the hepatocytes (30). In addition, S-(2chloroethy1)glutathione was identified in the bile of rats treated with BCE (13). To investigate the potential excretion of glutathione sulfur half-mustards from DHEtreated hepatocytes, we examined the effect of extracellular GSH on the formation of GEG. The presence of extracellular GSH resulted in a significant increase in the amount of GEG detected for DBE and BCE exposures. Extracellular GSH had little effect on HEG formation (data not shown). The finding that extracellular GSH had the greatest effect on GEG formation with DBE and BCE as compared with DCE may relate to their being better substrates for the GSTs, yielding greater levels of the S-(2-haloethyl)glutathionethan DCE. In vitro dipeptide, nucleoside, and GSH alkylation studies (14) have shown that cysteinyl thiol groups are alkylated at a rate more than 50-fold that of the N7-position of guanine or any other nucleic acid functional group. The extent of N7 adducts with calf thymus DNA (31) supports the evidence for relatively slow rates of nucleic acid alkylation as compared to cysteinyl thiol group alkylation by S-(2-haloethyl)glutathione. It became apparent in our metabolism studies that not all of the GSH depletion could be accounted for with the formation of HEG, CMG, and GEG. This was especially true for DBE, in which approximately 21.4 nmol of GSH/mL of cell suspension could not be accounted for after 1 h of incubation. The relative amounts of glutathione-containing metabolites formed and the investigations with 2-chloroacetaldehyde strongly suggest that the GST-catalyzed conjugation of DBE with GSH, which was responsible for formed S-(2-bromoethyl)glutathione, the majority of the GSH depletion and metabolite formation. Therefore, it is possible that the loss of GSH may be due to the alkylation of cellular macromolecules by S-(2-bromoethyl)glutathione. To investigate this further, we treated hepatocytes with [14C]DBEand measured the amount of covalent binding to hepatocyte protein. The covalent binding of [WIDBE occurred at the greatest rates under conditions of high intracellular GSH. In terms of accountability, the total amount of GSH present as GSH
Glutathione Metabolites of 1,2-Dihaloethanes
and glutathione-containingmetabolites, when added to the amount of covalent binding, agrees well with the total amount of GSH in controls. These data suggest an interesting relationship between the formation of glutathione sulfur half-mustards and covalent binding possibly of critical importance in DHE cell injury. We conclude that the GST-catalyzed conjugation of DHEs to GSH is very important in the overall utilization of GSH in DHE metabolism and toxicity of these solvents. Freshly isolated hepatocytes are being used to further investigate the role(s) of GSH and glutathione sulfur half-mustards in protein adduct formation and DHE hepatotoxicity.
Acknowledgment. We thank Marda Brown for isolation of rat hepatocytes and her excellent laboratory assistance. We especially thank Brian Arbogast and the Mass Spectrometry Core Unit of the EHS Center for mass spectral analyses. We thank Carolyn Knapp and Lynne Rogers for their assistance in preparing the manuscript. This research was supported by NIH Grants ES00040 and ES00210 from NIEHS. Registry No. DCE, 107-06-2;BCE, 107-04-0;DBE, 106-93-4; HEG, 28747-20-8; CMG, 10463-61-3; GEG, 128129-59-9; GSH, 70-18-8; 2-chloroacetic acid, 79-11-8; 2-chloroethanol, 107-07-3; 2-~hloroacetaldehyde,107-20-0.
References (1) IARC (1977) Ethylene dibromide. ZARC Monogr. 15, 195-209. (2) Gold, L. S. (1980) Human exposures to ethylene dichloride. In Banbury Report 5, Ethylene Dichloride: A Potential Health Risk? (Ames, B., Infante, P., and Reitz, R., Eds.) pp 209-225, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. (3) Fabricant, J. D., and Chalmers J. H. (1980) Evidence of the mutagenicity of ethylene dichloride and structurally related compounds. In Banbury Report 5,Ethylene Dichloride: A Potential Health Risk? (Ames, B., Infante, P., and Reitz, R., Eds.) pp 309-330, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. (4) Rannug, U. (1980) Genotoxicity of l,2-dibromoethane and 1,2dichloroethane. Mutat. Res. 76, 269-295. (5) Ward, J. M. (1980) The carcinogenicity of ethylene dichloride in Osborn-Mendel rata and B6C3F1 mice. In Banbury Report 5, Ethylene Dichloride: A Potential Health Risk? (Ames, B., Infante, P., and Reitz, R., Eds.) pp 35-49, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. (6) Weisburger, E. K. (1977)Carcinogenicity studies on halogenated hydrocarbons. Enoiron. Health Perspect. 21, 7-16. (7) World Health Organization (1987) Environmental Health Criteria '62;1,2-Dichloroethane,World Health Organization, Geneva. (8) Letz, G. A., Pond, S. A,, Osterloh, J. D., Wade, R. L., and Becker, C. E. (1984) Two fatalities after acute occupational exposure to ethylene dibromide. J. Am. Med. Assoc. 252, 2428-2431. (9) Yodaiken, R. E., and Babcock, J. R. (1973) 1,2-Dichloroethane poisoning. Arch. Enoiron. Health 26, 282-284. (10) Guengerich, F. P., Crawford, W. M., Domoradzki, J. Y., MacDonald, T. L., and Wantanbe, P. G. (1980) In vitro activation of 1,2-dichloroethaneby microsomal and cytosol enzymes. Toxicol. Appl. Pharmacol. 55,303-317. (11) Shih, T. W., and Hill, D. L. (1981) Metabolic activation of 1,2-dibromoethane by glutathione transferase and by microsomal mixed function oxidase: further evidence for formation of two reactive metabolites. Res. Commun. Chem. Pathol. Pharmacol. 33,449-461. (12) Rannug, U., Sundvall, A., and Ramel, C. (1978) The mutagenic effect of 1,2-dichloroethane on Salmonella typhimurium. I.
Chem. Res. Toxicol., Vol. 5, No. 3, 1992 391 Activation through conjugation with glutathione. Chem.-Bid. Interact. 20, 1-16. (13) Marchand, D. H., and Reed, D. J. (1989) Identification of the reactive glutathione conjugate S-(2-chloroethyl)glutathionein the bile of 1-bromo-2-chloroethane-treated rats by high-pressure liquid chromatography and precolumn derivatization with ophthalaldehyde. Chem. Res. Toricol. 2, 449-454. (14) Jean, P. A., and Reed, D. J. (1989) In vitro dipeptide, nucleoside, and glutathione alkylation by S-(2-chloroethyl)glutathione and S-(2-~hloroethyl)-~-cysteine. Chem. Res. Toxicol. 2,455-460. (15) Orrenius, S., and Jones, D. P. (1978) Functions of glutathione in drug metabolism. In Functions of Glutathione in Liver and Kidney (Sies, H., and Wendel, A., Eds.) pp 164-175, SpringerVerlag, Berlin, Heidelberg, and New York. (16) Moldeus, P., and Quanguan, J. (1987) Importance of the glutathione cycle in drug metabolism. Pharmacacol. Ther. 33,37-40. (17) Fariss, M. W., Brown, M. K., Schmitz, J. A., and Reed, D. J. (1985) Mechanism of chemical-induced toxicity 1. Use of a rapid centrifugation technique for the separation of viable and nonviable hepatocytes. Toxicol. Appl. Pharmacol. 79, 283-295. (18) Fariss, M. W., and Reed, D. J. (1987) High-performance liquid chromatography of thiols and disulfides: dinitrophenol derivatives. Methods Enzymol. 143, 101-109. (19) Nachtomi, E. (1970) The metabolism of ethylene dibromide in the rat. Biochem. Pharmacol. 19, 2853-2860. (20) Johnson, M. K. (1967) Metabolism of chloroethanol in the rat. Biochem. Pharmacol. 16, 185-199. (21) Johnson, M. K. (1965) The influence of some aliphatic compounds on the rat liver glutathione levels. Biochem. Pharmacol. 14, 1383-1385. (22) VanBladeren, P. J., Hoogeterp, J. J., Breimer, D. D., and van der Gen, A. (1981) The influence of disulfiram and other inhibitors of oxidative metabolism on the formation of 2-hydroxyethyl-mercapturic acid from l,2-dibromoethane by the rat. Biochem. Pharmacol. 30, 2983-2987. (23) Yllner, S. (1971) Metabolism of 1,2-di~hloroethane-'~C in the mouse. Acta Pharmacol. Toxicol. 30, 257-265. (24) Hill, D. L., Shih, T. W. Johnston, T. P., and Struck, R. F. (1978) Macromolecular binding and metabolism of the carcinogen 1,2dibromoethane. Cancer Res. 38, 2438-2447. (25) Cmarik, J. L., Inskeep, P. B., Meredith, M. J., Meyer, D. J., Ketterer, B., and Guengerich, F. P. (1990) Selectivity of rat and human glutathione S-transferases in activation of ethylene dibromide by glutathione conjugation and DNA binding and induction of unscheduled DNA synthesis in human hepatocytes. Cancer Res. 50, 2747-2752. (26) Inskeep, P. B., Koga, N., Cmarik, J. L. and Guengerich, F. P. (1986) Covalent binding of 1,2-dihaloethanesto DNA and stability of the major DNA adduct, S-[2-(N7-guanyl)ethyl]glutathione. Cancer Res. 46, 2839-2844. (27) Reed, D. J., and Foureman, G. L. (1986) A comparison of the alkylating capabilities of the cysteinyl and glutathionyl conjugates of 1,2-dichloroethane. In Biological Reactive Intermediates ZIZ (Kocsis, J. J., Jollow, D. J., Witmer, C. M., Nelson, J. O., and Snyder, R., Eds.) pp 469-475, Plenum Publishing Corp. (28) Sies, H. (1983) Reduced and oxidized glutathione efflux from liver. In Glutathione: Storage, Transport and Turnover in Mammals (Sakamoto, Y., Higashi, T., and Tateishi, N., Eds.) pp 63-90, Japan Scientific Societies Press, Tokyo. (29) Ormstad, K., and Orrenius, S. (1983) Metabolism of extracellular glutathione in small intestine and kidney. In Glutathione: Storage, Transport and Turnover in Mammals (Sakamoto, Y., Higashi, T., and Tateishi, N., Eds.) pp 107-128, Japan Scientific Societies Press, Tokyo. (30) Rannug, U., and Beije, B. (1979) The mutagenic effect of 1,2dichloroethane on Salmonella typhimurium. 11. Activation by the isolated perfused rat liver. Chem.-Biol.Interact. 24,265-285. (31) Humphreys, W. G., Kim, D., Cmarik, J. L., Shimada, T., and Guengerich, F. P. (1990) Comparison of the DNA-alkylating properties and mutagenic responses of a series of S42-haloethyl)-substituted cysteine and glutathione derivatives. Biochemistry 29, 10342-10350.
