Chem. Res. Toxicol. 1994, 7, 291-296
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Art ides Bioactivation of [13C]Dichloromethanein Mouse, Rat, and Human Liver Cytosol: 13C Nuclear Magnetic Resonance Spectroscopic Studies Mazzaz Hashmi,t Susanne Dechert,* Wolfgang Dekant,* and M. W. Anders'st Department of Pharmacology, University of Rochester, 601 Elmwood Avenue, Rochester, New York 14642, and Institut fur Toxikologie, Universitiit Wiirzburg, 0-8700 Wiirzburg, Versbacher Strasse 9, Federal Republic of Germany Received November 17, 1993'
Dichloromethane is tumorigenic in lungs and liver of B6C3F1 mice, but is not tumorigenic in rats or hamsters, and its toxicity is associated with glutathione-dependent bioactivation. The objective of the present studies was to investigate the glutathione-dependent bioactivation of [13Cldichloromethane in mouse, rat, and human liver cytosol and the fate of dichloromethanederived reactive intermediates with l3C NMR. [l3C1Formaldehyde hydrate, [13C]S-(hydroxymethyl)glutathione,and [l3C1methanolwere identified as metabolites of [13C]dichloromethane. [13C]S-(Chloromethyl)glutathione,a putative intermediate in the glutathione-dependent bioactivation of dichloromethane, or derived adducts were not observed. Moreover, no evidence for the formation of S,S'-methylenebis[glutathionel by reaction of glutathione and formaldehyde under physiological conditions was obtained, although methanol was observed as a product. S,S'-Methylenebis[glutathionel was, however, formed by reaction of glutathione and formaldehyde a t pH 1. S-(Chloromethy1)-N-acetyl-L-cysteinemethyl ester, a surrogate for S(chloromethyl)glutathione, was prone to hydrolysis. These results corroborate the finding that formaldehyde is a reactive intermediate formed during the glutathione-dependent bioactivation of dichloromethane that may be involved in the observed tumorigenicity of dichloromethane in susceptible species. The results also indicate that S-(chloromethy1)glutathione is an intermediate in the glutathione-dependent bioactivation of dichloromethane and may also play a role in its mutagenicity and carcinogenicity.
Introduction Dichloromethane is widely used as a solvent, in aerosol preparations, and as a paint stripper. The acute organotropic toxicity of dichloromethane is not remarkable (1). Dichloromethane is mutagenic in some, but not all, shortterm mutagenicity assays (for review, see ref 1). Dichloromethane is also mutagenic in Salmonella typhimurium (21, and the bacterial mutagenicity of dichloromethane is associated with its glutathione-dependent bioactivation (3-5). Indeed, the mutagenicity of dichloromethane is markedly enhanced in S. typhimurium TA1535 transfected with a plasmid vector containing the cDNA for rat glutathione S-transferase 5-5 (6). Long-term toxicity studies showed an elevated incidence of lung and liver tumors in mice, but not in rats of hamsters, exposed by inhalation to dichloromethane (7-9). The metabolism of dichloromethane has been extensively investigated. In rats and man, dichloromethane is metabolized to carbon monoxide (10,ll). The biotransformation of dichloromethane to carbon monoxide is catalyzed by the cytochromes P450 (12, 13). The glutathione S-transferase-catalyzed biotransformation of dichloromethane affords formaldehyde and formic acid t University of Rochester. t Univereitiit Wbzburg.
*Abstract published in Advance ACS Abstracts, March 15, 1994.
as products (14,151. Pharmacokinetic studies show that dichloromethane is metabolized by a high-affinity, lowcapacity oxidative pathway and by a low-affinity, highcapacity glutathione-dependentpathway (16). Moreover, additional studies implicate the glutathione-dependent pathway in the observed tumorigenicity of dichloromethane (16-18). Physiologically based pharmacokinetic studies show a correlation between tumor incidence and the amount of dichloromethane metabolized by the glutathione S-transferase pathway (19). In vitro studies with dichloromethane demonstrate that glutathionedependent biotransformation is higher in the mouse, a susceptible species to dichloromethane-induced tumor formation, than in the rat, which does not develop tumors after exposure to dichloromethane (17). The reactive intermediates associated with the glutathione-dependent tumorigenicity of dichloromethane have not been fully identified. DNA adducts have not been found in mice or rats exposed to dichloromethane, but dichloromethane-derived radioactivity is incorporated (20,21). into DNA after exposure to [14Cldichlor~methane The glutathione-dependent bioactivation of dichloromethane leads to the formation of two potentially genotoxic intermediates, S-(chloromethy1)glutathioneand formaldehyde (15). Chemical considerations indicate that S(chloromethy1)glutathione may alkylate tissue nucleophiles because its reactivity may be similar to that of
0893-228x/94/2707-0291$04.50/00 1994 American Chemical Society
292 Chem. Res. Toxicol., Vol. 7, No. 3, 1994
Hashmi et al.
GS'7
GS 1
-e
+
CIXH2G 2
&G S C Y r C l --------
GSCH,SG
7
CHQOH+ GSSG 8 s
Figure 1. Postulated mechanism for the glutathione-dependent bioactivation of dichloromethane. 1, glutathione; 2, dichloromethane; 3, S-(chloromethy1)glutathione;4, S,S'-methylenebis[glutathione];5, S-(hydroxymethy1)glutathione;6, formaldehyde; 7, formaldehyde hydrate; 8, methanol; 9, glutathione disulfide; GST, glutathione S-transferase.
chloromethyl ethers (22,23),which are carcinogenic (24, 25), although chloromethyl ethers are hydrolyzed more rapidly than chloromethyl sulfides (26). Formaldehyde is reported to be mutagenic in a range of in vitro test systems, including the human lymphoblast TK6 cell line (27)and S. typhimurium strains (28,29). A recent study failed to detect formaldehyde-induced mutagenicity in S. typhimurium TA1535 ( 6 ) . Formaldehyde participates in the formation of DNA-protein cross-links (30),and DNAprotein cross-links have been identified in mouse liver after exposure to [14Cldichloromethane (31). The objective of the present experiments was to explore the formation and fate of reactive intermediates of dichloromethane (Figure 1). 13C NMR was used to investigate the glutathione S-transferase-catalyzed biotransformation of ['3C]dichloromethane in mouse, rat, and human liver cytosol and to study the reactions of [l3C1formaldehyde with glutathione. 13CNMR was chosen as an analytical tool in an attempt to detect labile intermediates that may not be amenable to analysis by spectrophotometric or mass spectrometric methods. We report herein that the glutathione S-transferase-catalyzed biotransformation of dichloromethane (2,Figure 1)affords formaldehyde (6,Figure l),which is in equilibrium with formaldehyde hydrate (7, Figure l),as aproduct. Although S-(chloromethy1)glutathione (3,Figure 1) is a probable intermediate in the glutathione-dependent biotransformation of dichloromethane, no direct evidence for its formation was found. S-(Chloromethy1)glutathionewould, however, be expected to be short-lived and may escape detection by 13C NMR.
Experimental Section Chemicals. [WIDichloromethane (99atom % ) was obtained from Isotec, Inc. (Miamisburg, OH), and its 'H-decoupled 13C NMR spectrum in CDCl3 showed a singlet at 54.2 ppm. [l3C1Paraformaldehyde (99 atom % ) and a 20% aqueous solution of [13C]formaldehydewas purchased from MSD Isotopes (St. Louis, MO). All other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) or Aldrich Chemical Co. (Milwaukee, WI) in the highest purity available. Synthesis of [cLlorometLyJ-l~C]B(Chloromethyl)-Nacetyl-L-cysteine Methyl Ester. This compound was synthesized by the addition of [Wlparaformaldehyde (10 mmol, 0.3 g) to a cold (-15 "C)solution of N-acetyl-L-cysteine methyl ester (10 mmol, 1.63 g) under an argon atmosphere. Dry hydrogen chloride gas was passed through the reaction mixture until all paraformaldehyde had disappeared. The reaction mixture was extracted with dichloromethane. The organic layer was washed with a cold solution of sodium bicarbonate and dried over magnesium sulfate. After evaporation of dichloromethane under
reduced pressure, a colorless solid was obtained that was pure by lH NMR. The yield was 1.44 g (65%). 'H NMR (CDCls): 6 1.99 (s,3 H), 3.10 (dd, lH), 3.21 (dd, lH), 3.72 (s,3H), 4.57 (d, 1H),4.66 (d,lH),4.85 (m,lH),6.22 (8,1H). '*C NMR (CDCls): 6 23.5 (q, COCHs), 34.4 (t, CHCHzS), 49.6 (t, SCHZCl), 51.5 (9, OCH3). GC-MS (EI) m/z (re1 intensity): 190 (l), 176 ( 5 ) , 145 (111, 131 (271, 129 (11, 88 (1001, 86 (21). MS and NMR Analyses. Mass spectra were recorded with a Hewlett-Packard Model 5970 mass-selective detector coupled to a Hewlett-Packard Model 5890 gas chromatograph. 'H NMR and proton-coupled l3C NMR spectra used to characterize [chloromethyE-13C]S-(chloromethyl)-N-acetyl-~-cysteine methyl ester were recorded at 250 and 63 MHz, respectively,on a Bruker AC250 spectrometer. In all other experiments, proton-decoupled '3C NMR spectra were obtained with a General Electric QE-300 spectrometer equipped with a 5-mm multinuclear probe and operating at 75.60 MHz for 13C. The I3C NMR spectra were obtained with a spectral width of 5319 Hz and 32K data size. A 40" pulse width was used, and the recycle time was 3.08 s. The line-broadening parameter used in exponential multiplication of free-induction decay experiments was set to 3 Hz. The samples were incubated at 37 "C during the assay of metabolite formation, and the chemical shifts were referenced to ['SCIdichloromethane (6 = 54.2 ppm). Attached-proton-test experiments were used to produce decoupled '3C spectra with C-H multiplicity discrimination (32). The decoupler was kept on continuously except during the D1 delay after the P2 pulse. Pulse values for the attached-protontest experiments were as follows: P1,25.0ps; P2,7.50 ps; P3,12.5 ps. D5 and D6 delays were 1.50 and 20 ws, respectively. The recycle time was 3.20 s with a data size of 32K. The D1 delay was set to 7.50 ms so that methylene carbons produced positive signals and methyl and methine carbons produced negative signals. Preparation of Liver Cytosol. Male, B6C3F1 mice (24-26 g) and Fischer 344 rats (150-175 g) were purchased from Charles River Laboratories (Wilmington, MA). Human liver tissue was obtained from the University of Rochester Organ Procurement Program or from the Washington Regional Transplant Consortium (Fairfax, VA). Hepatic cytosolic fractions were prepared by homogenizing the minced tissue in a Dounce homogenizer in 100 mM phosphate buffer (pH 7.4). The homogenate was centrifuged at 9000gfor 20 min; the resultingsupernatant fraction was centrifuged at lOOOOOg for 60 min to yield the cytosolic fraction. The cytosol was dialyzed (Spectrapor 3 tubing, Spectrum Medical Industries, 3500 molecular weight cutoff) in 100 mM phosphate buffer (pH 7.4) for at least 24 h at 4 OC. Protein concentrations were determined by the method of Bradford (33) with bovine serum albumin as the standard. Sample Preparation for NMR Analyses. Incubation mixtures were prepared in 5-mm diameter NMR tubes and contained 20 mM phosphate buffer (pH 7.4), 10mMglutathione, 72 pmol of [13C]dichloromethane [added as a liquid with a cold (4 "C) gas-tight syringe], 15% DzO, and 8 mg of cytosolicprotein in a total volume of 1mL. The reactions were started by addition of cytosol, and the acquisition of NMR transients was started within 1 min after addition of cytosol. Samples for attached-proton-test experiments contained 20 mMphosphate buffer (pH 7.4), 10mM glutathione, 50 mM ['VIformaldehyde, and 15% DzO in a total volume of 1 mL. Biotransformation of Dichloromethane to Formaldehyde. Incubation mixtures contained 20 mM phosphate buffer (pH 7.4), 10 mM glutathione, 72 pmol of dichloromethane, and 8 mg of cytosolic protein in a total volume of 3 mL. The reaction mixtures were incubated at 37 "C in an atmosphere of air. After 15, 30, and 45 min of incubation, formaldehyde formation was quantified by the method of Nash (34).
Results Biotransformation of Dichloromethane to Formaldehyde. Incubation of mouse, rat, and human hepatic
Chem. Res. Toxicol., Vol. 7, No. 3, 1994 293
Bioactiuation of [13C]Dichloromethane c
A
B
Time (min)
Figure 2. Time-dependent formation of formaldehyde 6 from dichloromethane 2 in the presence of hepatic cytosolic protein. Mouse (B), rat (A),or human (e)liver cytosol was incubated with glutathione and dichloromethane, and formaldehyde formation was quantified,as described in the ExperimentalSection. Data are shown as mean f SD for three experiments.
cytosol with dichloromethane in the presence of glutathione showed a time-dependent increase in formaldehyde formation (Figure 2). The rate of formaldehyde formation was highest in mouse cytosol followed by rat and human liver cytosol (Figure 2). This confirms that formaldehyde is a metabolite of dichloromethane and establishesthe incubation conditions for 13CNMR studies on the glutathione-dependent biotransformation of dichloromethane. 1% NMR Analyses with Liver Cytosol. When mouse liver cytosol was incubated with [l3C1dichloromethane and the 13C NMR spectrum was recorded, several new resonances appeared. After 45 min of incubation, resonances at 82.3,65.6,49.3,and 43.6 ppm were seen (Figure 3 0 . No resonances were detected in incubation mixtures that lacked glutathione (Figure 3A) or cytosol (Figure 3B). The most intense resonance in the spectrum at 65.6 ppm was assigned to S-(hydroxymethy1)glutathione (5, Figure l ) , in accordance with previous reports (35,361. This signal was in equilibrium with the signal at 82.3 ppm, which was assigned to formaldehyde hydrate (7, Figure 1) (35,361. The resonances at 49.3 and 43.6 ppm were consistently observed. After incubation of [W l dichloromethane with rat liver cytosol, resonances were detected (Figure 4C)with chemical shifts similar to those found in the mouse, indicating the formation of similar metabolites. The intensities of the resonances were lower in the rat than in the mouse. Again, no resonances were detected in incubation mixtures that lacked glutathione (Figure 4A) or cytosol (Figure 4B). Under similar conditions, detectable resonances were not observed after incubation of [13Cldichlor~methane and glutathione with human liver cytosol. Reaction of Glutathione with [WIFormaldehyde. The reaction of glutathione (10 mM) and P3C1formaldehyde (50 mM), at pH 7.4 in the absence of cytosol, gave resonances at 82.3, 65.6, 61.0, 60.5, 53.5, 49.3, and 43.6 ppm (Figure 5). The resonances at 61.0 and 60.5 ppm were assigned to bicyclic adduct 10 (Figure 61, which has been observed previously (35). The resonance at 53.5 ppm was assigned to cyclic adduct 11 (Figure 6), as previously reported (35). Attached-proton-test experiments were used to identify the weak resonance at 49.3 ppm (Figure 5 ) . When a
k
I
C
I
I " " l " " I " " I " " l " " I " " l " " l
100
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60
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60
50
40
30 PPM
Figure 3. 13C NMR spectra after incubation of [Wldichloromethane (A) in the absence of glutathione, (B)in the absence of mouse hepatic cytosol,and (C)in the presence of mouse hepatic cytosoland glutathione. The inset shows a region of the spectrum that has been enlarged 10-fold. The observed resonances are assigned to structures shown in Figure 1: 7, 82.3 ppm; 5, 65.6 ppm; 8,49.3ppm;unidentified product, 43.6 ppm. These spectra represent the accumulation of 850 scans (45min).
reaction mixture containing [l3C1formaldehyde and glutathione in phosphate buffer (pH 7.4) was incubated in the NMR probe, an intense signal at 65.6 ppm appeared. New resonances gradually appeared as the resonance at 65.6 ppm decreased in intensity. Signals at 61.0 and 60.5 ppm appeared simultaneously, and the former was slightly more intense than the latter and were assigned as indicated above. The signal at 53.5 ppm was assigned to the cyclic adduct 11,as indicated above. The intense resonances at 49.3 and 43.6 ppm were also seen in the attached-protontest experiments (Figure 7). Two weak resonances at 51.4 and 72.4 ppm were observed, but not identified. All resonances,except the resonance at 49.3 ppm, gave positive signals, indicating the presence of methylene carbons. The resonance at 49.3 ppm appeared as a negative signal in the attached-proton-test experiments, which identifies the C-H multiplicity as a methyl carbon. The intensity of the signal at 49.3 ppm increased after the addition of 20 p L of methanol in a one-dimensional I3C NMR experiment (data not shown). Reactivity of [~hlorometbyl-~ W]S-(Chloromethy1)N-acetyl-L-cysteineMethyl Ester. This compound was synthesized as a surrogate to examine the potential reactivity and stability of S-(chloromethy1)glutathione (3, Figure l), a putative intermediate formed during the bioactivation of dichloromethane. When dissolved in 20 mM phosphate buffer (pH 7.4), [chloromethyl-13C]S(chloromethy1)-N-acetyl-L-cysteine methyl ester was rapidly hydrolyzed to give signals at 65.6 ppm, assigned to [hydroxymethyZ-l3C]S-(hydroxymethy1)-N-acetyl-L-cys-
Hashmi et al.
294 Chem. Res. Toxicol., Vol. 7, No. 3, 1994 A
....-.-.-..-ur'k B
/ " " 1 " " I " " I " " 1 " " I " " / " " I
100
90
80
70
60
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40
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30 PPM
Figure 4. 13C NMR spectra after incubation of [Wldichloromethane (A) in the absence of glutathione, (B) in the absence of rat hepatic cytosol, and (C) in the presence of rat hepatic cytosol and glutathione. The inset shows resonances that are enlarged 10-fold. The observed resonances are assigned to structures shown in F' e 1: 7, 82.3 ppm; 5,65.6 ppm; 8,49.3 ppm; unidentified pr O uct, Y 43.6 ppm. These spectra represent the accumulation of 850 scans (45 min).
100
40
50
60
70
80
Figure 7. 13CNMR attached-proton-test spectrum after incubation of [Wlformaldehyde (50mM) with glutathione (10 mM) at pH 7.4. The spectrum (1900 scans) was recorded after 100 min of incubation. The inset shows a region of the spectrum that has been enlarged 10-fold. The observed resonances are assigned to structures shown in Figures 1and 4 7,82.3 ppm; 5, 65.6 ppm; 10, 61.0 and 60.5 ppm; 11, 53.5 ppm; 8, 49.3 ppm (negative signal); unidentified product, 43.6 ppm. In the attached-proton-test spectrum, the positive signals indicate resonances from carbon atoms attached to an even (0,2)number of protons, whereas negative signals indicate resonances from carbon atoms attached to an odd (1,3) number of protons (32).
C
100
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90
80
70
60
50
40
30 PPM
Figure 5. 13C NMR spectrum after incubation of glutathione (10 mM) and ['%]formaldehyde (50 mM) at pH 7.4. The spectrum (650 scans) was recorded after 34 min of incubation. The observed resonances are assigned to structures shown in Figures 1and 4: 7,82.3 ppm; 6,65.6 ppm; 10,61.0 and 60.5 ppm; 11, 53.5 ppm; 8, 49.3 ppm; unidentified product, 43.6 ppm.
100
90
80
70
60
50
40
30
PPM
Figure 8. 13C NMR spectrum after incubation of [Wlformaldehyde (50 mM) with glutathione (100 mM) at pH 1. The spectrum (96 scans) was recorded after 5 min of incubation. The observed resonances are assigned to structures shown in Figure 1: 5, 65.6 ppm; 4, 35.6 ppm. 65.6 ppm (Figure 8). Preliminary kinetic studies with S-(chloromethy1)-N-acetyl-L-cysteine methyl ester gave a half-life of about 4 s at 0 "C in 100 mM phosphate buffer (pH 7.4).
Discussion
T h e formation of formaldehyde after incubation of dichloromethane with mouse, rat, and human hepatic cytosol in the presence of glutathione confirms previous observations that dichloromethane is biotransformed to formaldehyde (14,15,17). The rank order (mouse > rat > human) of the biotransformation of dichloromethane to formaldehyde by hepatic cytosols found in the present experiments agrees with previous reports (17). Formal11 dehyde is mutagenic in a range of test systems (27-291, and its formation may be involved in the observed 10 mutagenicity and tumorigenicity of dichloromethane. Figure 6. Structures of cyclic adducts of formaldehyde and Recent in vivo studies indicate that the potential tumglutathione, as reported by Naylor et al. (35). Bicyclic adduct origenicity may be associated with the formation of DNA1 0 61.0 and 60.5 ppm. Cyclic adduct 11: 53.5 ppm. protein cross-links of formaldehyde in B6C3F1 mice (31). In the present studies, l3C N M R was used to detect and teine methyl ester, at 82.3 ppm, assigned to [l3C1formidentify metabolites of dichloromethane formed in incualdehyde hydrate, and at 35.6 ppm. T h e weak signal a t 35.6 ppm was assigned to [methylene-l3CIS,S'-methyl- bation mixtures. The first resonance (65.6 ppm) that appeared when [ W I dichloromethane was incubated with enebis[N-acetyl-L-cysteinemethyl ester]. Synthetic [meglutathione in the presence of mouse and rat liver cytosol thylene-l~CIS,S'-methylenebis[glutathionel, prepared by was assigned to S-(hydroxymethy1)glutathione (5, Figure the reaction of glutathione (100 mM) with [13C]formaldehyde (50 mM) a t p H 1(33,gave resonances a t 35.6 and 1). This product may be formed by the hydrolysis of
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Bioactivation of [*3C]Dichloromethane
S-(chloromethy1)glutathione (3, Figure l), the initial product of the glutathione S-transferase-catalyzed attack of glutathione on dichloromethane. S-(Hydroxymethy1)glutathione is the hemithioacetal of glutathione and formaldehyde and exists in equilibrium with glutathione and formaldehyde. The equilibrium between Whydroxymethy1)glutathione and glutathione and formaldehyde (5 s 1+ 6, Figure 1)has been well characterized and is highly sensitive to pH and to the relative concentrations of formaldehyde and glutathione (35, 36, 38). Furthermore, the attached-proton-test experiments and coresonance with methanol show that S-(hydroxymethy1)glutathione may afford methanol as a product (8, Figure 1). Although the reaction was not investigated in detail, the attack of glutathione on S-(hydroxymethy1)glutathione may give methanol and glutathione disulfide (1 + 5 8 + 9, Figure 1)as products. Analogous reactions have been reported: the attack of glutathione on S-(2-chloroethyl)glutathione gives ethene and glutathione disulfide as products (39). Similarly, the enzyme-catalyzed attack of glutathione on S-(2’,4’-dichlorophenacyl)glutathionegives glutathione disulfide and 2’,4’-dichloroacetophenoneas products (40, 41). The failure to detect a resonance assignable to [chloromethyZ-13ClS-(chloromethyl)glutathione or its derived adducts in the in vitro experiments indicates that this intermediate is labile. The analog S-(chloromethy1)-Nacetyl-L-cysteinemethyl ester undergoes rapid hydrolysis at pH 7.4 to form S-(hydroxymethy1)-N-acetyl-L-cysteine methyl ester and formaldehyde hydrate as major products and S,S’-methylenebis [N-acetyl-L-cysteinemethyl ester] as a minor product. This indicates that the chloromethyl sulfide is hydrolyzed to S-(hydroxymethy1)-N-acetyl+ cysteine methyl ester and thence to formaldehyde and N-acetyl-L-cysteine methyl ester; the reaction of S(chloromethy1)-N-acetyl-L-cysteine methyl ester with the N-acetyl-L-cysteine methyl ester thus formed could account for the observed formation of S,S’-methylenebis[N-acetyl-L-cysteine methyl ester]. Studies on the reactivity of S-(chloromethyl)-N-acetyl-L-cysteinemethyl ester indicate that, although S-(chloromethy1)glutathionewould be labile, it may bind covalently with proteins or DNA. The analog S-(l-acetoxymethy1)glutathionereacts with 2’-deoxyguanosine to give S- [l-(W-deoxyguanosiny1)methyllglutathione (6). The formation of the S,S’-methylenebis[glutathionel (4, Figure 11,by reaction of glutathione with formaldehyde (5 + 7 4, Figure 1) was not observed when dichloromethane was incubated in the presence of glutathione and cytosol, which indicates that the formation of 4 is not favored under physiological conditions. Indeed, the chemical synthesis of djenkolic acid (S,S’-methylenebis[~-cysteinel)requires strongly acidic conditions (37). Moreover, in the present studies, S,S’-methylenebis[glutathione] was formed when glutathione and formaldehyde were incubated under acidic conditions (Figure 8). These observations indicate that under physiological conditions neither glutathione and formaldehyde nor glutathione and S-(chloromethy1)glutathionereact to form S,S’-methylenebis[glutathionel in quantities detectable by 13C NMR. The unidentified resonance at 43.6 ppm has been observed previously (35). This resonance may be assignable to a cross-linked methylene carbon arising from the intermolecular condensation of S-(hydroxymethy1)glutathione and one of the amide nitrogens of glutathione.
-
-
This resonance was not detected when [l3C1formaldehyde was incubated with N-acetyl-L-cysteine and L-glutamic acid (data not shown), indicating that the intermediate does not react with amino group of glutathione. The amino groups of adenosine and guanosine derivatives react with formaldehyde and thiols to form N - [(alkylthio)methyll derivatives of nucleotides (42-44). In summary, the present findings show that [13C]formaldehyde (6,Figure l),[Wlformaldehyde hydrate (7, Figure 11, and the glutathione hemithioacetal [l3C1S-(hydroxymethy1)glutathione (5, Figure l), are metabolites of [13Cldichloromethane. The glutathione S-transferase-catalyzed reaction of glutathione with dichloromethane would be expected to afford S(chloromethy1)glutathione as an intermediate, but the apparent lability of this intermediate prevented its detection in the present experiments. Studies with the analog S-(chloromethy1)N-acetyl-L-cysteinemethyl ester showed that this chloromethyl sulfide is hydrolytically labile, and its lability apparently precluded its detection by 13C NMR. [W]Methanol (8, Figure 11, which apparently arises by attack of glutathione on S-(hydroxymethyl)glutathione,was also detected as a minor metabolite. These results are in agreement with the concept that formaldehyde is a toxicologically significant metabolite formed during the glutathione-dependent bioactivation of dichloromethane and may play a role in the tumorigenicity of dichloromethane in susceptible species. Further studies are needed to explore the formation and reactivity of S-(chloromethy1)glutathioneand its possible role in the mutagenicity and tumorigenicity of dichloromethane.
Acknowledgment. The authors thank Sandra E. Morgan for her assistance in preparing the manuscript. This research was supported by National Institute of Environmental Health Sciences Grant E503127 (M.W.A.), by the Deutsche Forschungsgemeinschaft Sonderforschungsbereich 76 (W.D.), and by NATO Grant 901032 (M.W.A., W.D.). References (1) International Agency for Research on Cancer (1986) in IARC Monographs on the Eualuation of the Carcinogenic Risk of Chemicals to Humans, Volume31,Some HalogenatedHydrocarbons and Pesticide Exposures, pp 43-85, IARC, Lyon, France. (2) Jongen, W. M. F., Alink, G. M., andKoeman, J. H. (1978) Mutagenic effect of dichloromethane on Salmonella typhimurium. Mutat. Res. 56, 245-248. (3) Jongen, W. M. F., Harmsen, E. G. M., Alink, G. M., and Koeman, J. H. (1982) The effect of glutathione conjugation and microsomal oxidation on the mutagenicity of dichloromethane in S.typhimurium. Mutat. Res. 95, 183-189. (4) Green, T. (1983) The metabolic activation of dichloromethane and chlorofluoromethanein a bacterial mutation assay using Salmonella typhimurium. Mutat. Res. 118, 277-288. (5) Dillon, D., Edwards, I., Combes, R., McConville, M., and Zeiger, E. (1992)The role of glutathione in the bacterial mutagenicityof vapour phase dichloromethane. Enuiron. Mol.Mutagen. 20, 211-217. (6) Thier, R.,Taylor, J. B., Pemble, S. E., Humphreys, W. G., Persmark, M., Ketterer, B., and Guengerich, F. P. (1993) Expression of mammalian glutathione S-transferase 5-5 in Salmonella typhimurium TA1535 leads to base-pair mutations upon exposure to dihalomethanes. h o c . Natl. Acad. Sci. U.S.A. 90,8576-8580. (7) National Toxicology Program (1986) The toxicology and carcinogenesis of dichloromethane (methylene chloride) in F344/N rata and BGC3Flmice (inhalation studies). N T P Technical Report 306. Final Report. (8) Mennear, J. H., McConnell, E. E., Huff, J. E., Renne, R. A,, and Giddens, E. (1988) Inhalation toxicology and carcinogenesisstudies of methylene chloride (dichloromethane)in F344/N rata and B6C3F1 mice. Ann. N.Y. Acad. Sci. 534, 343-351.
296 Chem. Res. Toxicol., Vol. 7, No. 3, 1994 (9) Burek, J. D., Nitachke, K. D., Bell, T. J., Wackerle, D. L., Childs, R. C.. Beuer, J. E., Dittenber. D. A., R ~ ~ DL.YW., , and McKenna, J. J. (198;1)Methylene chloride: A 2-year inhalation toxicology and oncogenicity study in rats and hamsters. Fundam. Appl. Toxicol. 4,3c-47. Kubic,V. L., Anders,M. W.,Engel,R. R.,Barlow, C. H.,andCaughey, W. S. (1974)Metabolism of dihalomethanes to carbon monoxide. I. In vivo studies. Drug Metab. Dispos. 2,53-57. Stewart, R. D., Fisher, T. N., Hosko, M. J., Peterson, J. E., Baretta, E. D., and Dodd, H. C. (1972)Experimental human exposure to methylene chloride. Arch. Environ. Health 25,342-348. Kubic, V. L., and Anders, M. W. (1975)Metabolism of dihalomethanes to carbon monoxide. 11. In vitro studies. Drug Metab. Dispos. 3, 104-112. Kubic, V. L., and Anders, M. W. (1978)Metabolism of dihalomethanes to carbon monoxide. 111. Studies on the mechanism of the reaction. Biochem. Pharmacol. 27,2349-2355. Ahmed, A. E., and Anders, M. W. (1976)Metabolism of dihalomethanes to formaldehyde and inorganic halide. I. In vitro studies. Drug Metab. Dispos. 4,357-361. Ahmed, A. E., and Anders, M. W. (1978)Metabolism of dihalomethanes to formaldehyde and inorganic halide. 11. Studies on the mechanism of the reaction. Biochem. Pharmacol. 27,2021-2025. Gargas, M. L.,Clewell, H. J., 111, and Andersen, M. E. (1986) Metabolism of inhaled dichloromethanesin vivo: Differentiation of kinetic constants for two independent pathways. Toxicol. Appl. Pharmacol. 82,211-223. Reitz, R. H., Mendrala, A. L., and Guengerich, F. P. (1989)In vitro metabolism of methylene chloride in human and animal tissues: Use in physiologically based pharmacokinetic models. Toxicol. Appl. Pharmacol. 97,230-246. Green, T.(1989)A biological data base for methylene chloride risk assessment. In Biologically-Based Methods for Cancer Risk Assessment (Travis, C. C., Ed.) pp 289-300, Plenum, New York. Andersen, M. E., Clewell, H. J., 111, Gargas, M. L., Smith, F. A., and Reitz, R. H. (1987)Physiologically based pharmacokineticsand the risk assessment process for methylene chloride. Toxicol. Appl. Pharmacol. 87, 185-205. Green, T., Provan, W. M., Collinge, D. C., and Guest, A. E. (1988) Macromolecular interactions of inhaled methylene chloride in rata and mice. Toxicol. Appl. Pharmacol. 93,1-10. Ottenwader, H.,and Peter, H. (1989) DNA binding assay of methylene chloride in rats and mice. Arch. Toxicol. 63,162-163. Bohme, H., Fischer, H., and Frank, R. (1949) Preparation and properties of a-halogenated thioethers. Ann. Chem. 563,54-72. Tou, J. C., and Kallos, G. J. (1974)Kinetic study of the stabilities of chloromethyl methyl ether and bis(chloromethy1)ether in humid air. Anal. Chem. 46,1866-1869. van Duuren, B. L., Sivak, A,, Goldschmidt, B. M., Katz, C., and Melchionne,S. (1969)Carcinogenicityof halo-ethers. J.Nut. Cancer Inst. 43,481-486. DeFonso, L. R., and Kelton, S. C., Jr. (1976)Lung cancer following exposure to chloromethyl methyl ether. Arch. Enuiron. Health 31, 125-130. Bohme, H. (1941)Influence of 0 and S atoms in the a-position on the hydrolysis velocityof the carbon-halogen bond. Chem. Ber. 74, 248-256. Goldmacher, V. S., and Thilly, W. G. (1983) Formaldehyde is mutagenic for cultured human cells. Mutat. Res. 116,417-422. Levin, D. E.,Hollstein, M., Christman, M. F., Schwiers, E. A., and Ames, B. N. (1982)A New Salmonella tester strain (TA102) with
Hashmi et al. A-T base pairs at the site of mutation detecta oxidative mutagens. Proc. Natl. Acad. Sci. U.S.A. 79. 7445-7449. (29) Takahashi, K., Morita, T., and Kawazoe, Y. (1985)Mutagenic characteristics of formaldehyde on bacterial systems. Mutat. Res. 156,153-161. (30) Solomon, M. J., and Varshavsky, A. (1985)Formaldehyde-mediated DNA-protein crosslinking: A probe for in uiuo chromatinstructures. Proc. Natl. Acad. Sci. U.S.A. 82,6470-6474. (31) Casanova,M., Deyo, D. F., and Heck, H. d’A. (1992)Dichloromethane (methylene chloride): Metabolism to formaldehyde and formation of DNA-protein cross links in B6C3F1 mice and Syrian Golden hamsters. Toxicol. Appl. Pharmacol. 114,162-165. (32)Patt,S.L., andshoolery, J. N. (1982)Attachedprotontestforcarbon13 NMR. J. Magn. Reson. 46,535-539. (33)Bradford,M. (1976)Arapidandsensitivemethodforthequantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal. Biochem. 72,248-254. (34) Nash, T. (1953)The colorimetric estimation of formaldehyde by means of the Hantzch reaction. Biochem. J. 55,416-421. (35) Naylor, S., Mason, R. P., Sanders, J. K. M., Williams, D. H., and Moneti, G.(1988)Formaldehyde adducta of glutathione. Structure elucidation by two-dimensional NMR spectroscopy and fast-atombombardment tandem mass spectrometry. Biochem. J. 249,573579. (36) Mason, R. P., Sanders, J. K. M., Crawford, A., and Hunter, B. K. (1986)Formaldehyde metabolism by Escherichia coli. Detection by in vivo 13C NMR spectroscopy of S-(hydroxymethy1)glutathione as a transient intracellular intermediate. Biochemistry 25,45044507. (37) Armstrong, M. D., and du Vigneaud, V. (1947)A new synthesis of djenkolic acid. J. Biol. Chem. 168,373-377. (38)Uotila, L., and Koiwalo, M. (1974)Formaldehyde dehydrogenase from human liver. Purification, properties, and evidence for the formation of glutathione thiol esters by the enzyme. J.Biol. Chem. 249,7653-7663. (39) Livesey, J. C., Anders, M. W., Langvardt, P. W., Putzig, C. L., and Reitz, R. H. (1982)Stereochemistry of the glutathione-dependent biotransformation of vicinal-dihaloalkaneato alkenes. Drug. Metab. Dispos. 10,201-204. (40) Brundin, A., Ratnayake, J. H., Sunram, J. M., and Anders, M. W. (1982)Glutathione-dependent reductive dehalogenation of 2,2’,4’trichloroacetophenone to 2’,4’-dichloroacetophenone. Biochem. Pharmacol. 31,3885-3890. (41) Kitada, M., McLenithan, J. C., and Andera, M. W. (1985)Purification and characterization of S-phenacylglutathionereductase from rat liver. J.Biol. Chem. 260, 11749-11754. (42) Yamazaki, Y., and Suzuki, H. (1978)A new method of chemical modification of Ne-amino group in adenine nucleotides with formaldehyde and a thiol and ita application to preparing immobilized ADP and ATP. Eur. J. Biochem. 92,197-207. (43) Yamazaki, Y., Suzuki, H., Kamibayhashi, A., Watanabe, N., and Takahashi, N. (1979)W-Alkylthiomethyl and W-arylthiomethyl derivatives of adenosine and their cytokinin activity. Agric. Biol. Chem. 43, 1945-1950. (44) Bridson, P. K., and Reese, C. B. (1979)A novel method for the methylation of heterocyclic amino groups. Conversion of guanosine into ita 2-N-methyl- and 2-N,2-N-dimethyl derivatives. Bioorg. Chem. 8, 339-349.
