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0090-9556/97/2504-0508-0515$02.00/0
DRUG METABOLISM AND DISPOSITION
Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
Vol. 25, No. 4

Urinary Thiodiacetic Acid
A Selective Biomarker for the Cytochrome P450-Catalyzed Oxidation of 1,2-Dibromoethane in the Rat

Lars W. Wormhoudt, Jan N. M. Commandeur, Jan H. T. M. Ploemen, Ruben S. Abdoelgafoer, Arman Makansi, Peter J. van Bladeren, and Nico P. E. Vermeulen

Leiden Amsterdam Center for Drug Research (L.W.W., J.N.M.C., R.S.A., A.M., N.P.E.V.), Department of Pharmacochemistry, Division of Molecular Toxicology, Vrije Universiteit; and Division of Toxicology (J.H.T.M.P., P.J.v.B.), TNO Nutrition and Food Research Institute

    Abstract
Abstract
Introduction
Results
Discussion
References

1,2-Dibromoethane (1,2-DBE) is a carcinogenic compound that is metabolized both by cytochrome P450 (P450) and glutathione S-transferase (GST) enzymes, and that has been used by us as a model compound to study interindividual variability in biotransformation reactions. In this study, the excretion of thiodiacetic acid (TDA) and S-(2-hydroxyethyl)-N-acetyl-l-cysteine (2-HEMA) were measured in the urine of rats dosed with 1,2-DBE, and experiments were performed to investigate to what extent P450 and GST enzymes contribute to the formation of TDA. To this end, CYP2E1, the main P450 isoenzyme catalyzing the oxidation of 1,2-DBE, was inhibited using disulfiram and diallylsulfide. Significant inhibition of CYP2E1, as confirmed by inhibition of the hydroxylation of chlorzoxazone, as well as inhibition of the formation of TDA from 1,2-DBE, was observed upon pretreatment of rats with these inhibitors, indicating that the P450-catalyzed oxidation of 1,2-DBE plays the major role in the TDA formation. No significant excretion of TDA was observed after administration of intermediate products of the GST pathway [i.e. S-(2-hydroxyethyl)glutathione and 2-HEMA], indicating that the GST-catalyzed metabolism of 1,2-DBE does not contribute to a significant extent to the formation of TDA. The results of this study show that TDA is specifically formed by P450 metabolites of 1,2-DBE, whereas the conjugation of 1,2-DBE to glutathione by GST enzymes does not contribute to the formation of TDA. TDA, excreted in urine, may thus be used as a biomarker of exposure to 1,2-DBE selectively reflecting the P450-catalyzed oxidation. In addition to 2-HEMA and S-[2-(N7-guanyl)ethyl]-N-acetyl-l-cysteine, TDA may be a valuable tool for biomonitoring and mechanistic studies into the metabolism and toxicity of 1,2-DBE.

    Introduction
Abstract
Introduction
Results
Discussion
References

1,2-DBE1, is used as an additive in leaded gasoline and as a soil fumigant (for a review, see ref. 1). It has been shown to be genotoxic in a variety of test systems (2-7) and is suspected to be a carcinogen in humans, although convincing evidence is still lacking (8, 9). The metabolism of 1,2-DBE is catalyzed both by P450 and GST enzymes. Upon GST-catalyzed metabolism, a reactive episulfonium ion is formed, which is able to react with the N7-position in guanine DNA bases and is probably involved in the genotoxic effects of 1,2-DBE (3, 10). Furthermore, the GST-catalyzed metabolism of 1,2-DBE has been shown to lead to embryotoxicity in an in vitro study (11). Upon oxidation of 1,2-DBE by P450, the reactive 2-BA is formed, which may bind to cellular proteins or, alternatively, conjugate to GSH and enter the mercapturic acid pathway (12-15). Substantial evidence has been presented indicating that human CYP2E1 is the major P450 isoenzyme that catalyzes oxidation of 1,2-DBE (16-18).

Two important urinary metabolites of 1,2-DBE are 2-HEMA (3, 13, 19) and 2-GEMA (20) (fig. 1). 2-HEMA is ultimately formed from reactive intermediates of both the P450- and GST-catalyzed 1,2-DBE metabolism, whereas 2-GEMA is formed as a degradation product after 1,2-DBE has been metabolized selectively by GST isoenzymes and the episulfonium ion thus formed has reacted with guanine DNA bases at the N7-position.


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  		Fig. 1.   Schematic representation of the metabolism of 1,2-DBE to products that can be excreted in urine.

The biotransformation of 1,2-DBE to the reactive intermediate 2-BA and a reactive episulfonium ion by P450 and GST enzymes, respectively, is shown. Also shown are three products, ultimately derived from these reactive intermediates, that can be excreted in urine. As can be seen, 2-HEMA is derived from both reactive intermediates, whereas 2-GEMA is formed selectively by the GST-catalyzed route of metabolism. Measurement of TDA as a biomarker for 1,2-DBE, possibly selectively reflecting the P450-catalyzed route of metabolism, is the subject of this study.

Urinary mercapturic acids have been proposed as useful tools for biological monitoring and mechanistic studies into the metabolism of electrophilic chemicals (14, 21). For example, it has been shown, by comparing urinary excretion of differentially deuterated 2-HEMA derived from D4-1,2-DBE, that the ratio of oxidation of 1,2-DBE vs. conjugation to GSH is ~4:1 in the rat (13). Because 2-HEMA only reflects the total internal dose of 1,2-DBE (i.e. the amount of 1,2-DBE metabolized by both P450 and GST enzymes), and 2-GEMA reflects the amount of 1,2-DBE metabolized selectively by GST enzymes, a urinary metabolite that reflects the amount of 1,2-DBE metabolized selectively by P450 enzymes is still lacking. In principle, TDA might be used as an alternative to 2-HEMA as a biomarker (22). Recently, TDA was also measured as a metabolite of 1,2-DCE (23). If TDA would be specifically formed from intermediate products of the P450-catalyzed oxidation of 1,2-DBE, it might thus be useful as a biomarker selectively reflecting the amount of 1,2-DBE metabolized by P450 (fig. 1).

The aim of the present study therefore was to investigate whether TDA is a urinary metabolite of 1,2-DBE and whether or not it can be used as a biomarker selectively reflecting the P450-catalyzed oxidation of 1,2-DBE.

Materials and Methods

Animals. The animals used were male Wistar Unilever rats that were obtained from Harlan (Zeist, the Netherlands). Rats were housed in groups for at least 1 week upon arrival in a temperature (21°C-22°C)- and humidity (60-65%)-controlled room with a light/dark cycle of 12 hr. Animals had free access to normal tap water and food [standard laboratory diet obtained from Hope Farms (Woerden, the Netherlands)] during this period. Experiments in which metabolism cages were used were conducted in a similar housing facility. At the time the experiments were conducted, the rats had body weights between 240 and 260 g.

Materials. 1,2-DBE was obtained from EGA Chemie (Steinheim, Germany). 2-Benzoxazolinone was obtained from Fluka (Buchs, Switzerland). BEMA, 2-HEMA, DSF, and DAS were obtained from Acros Chimica (Geel, Belgium). GSH was obtained from Boehringer (Mannheim, Germany). 2-Bromoethanol and CZX were obtained from Sigma Chemical Co. (St. Louis, MO). 6-OH-CZX was obtained from Research Biochemicals International (Natick, MA). TDA and 3-(trimethylsilyl)-propionic acid sodium salt were obtained from Merck (Darmstadt, Germany). Arachidis oil was obtained from the pharmacy of the Vrije Universteit Hospital (Amsterdam, the Netherlands). Ethanol, methanol, and ethyl acetate were obtained from Baker (Deventer, the Netherlands). HCl was obtained from Riedel-deHaën (Seelze, Germany).

2-HEG. GSH (5 g, 16.3 mmol) was dissolved in a sodium methanolate solution prepared by adding 1.12 g (48.9 mmol) sodium in 25 ml of methanol. After stirring at room temperature for 10 min, 2-bromoethanol (5.4 g, 43.2 mmol) was added dropwise, and 5 ml of water was added to dissolve all reagents completely. After 1 hr, the reaction was completed as judged by TLC. The reaction mixture was neutralized with HCl, and solvents were removed using a rotary evaporator. The identity of the compound was confirmed by NMR and MS methods. NMR spectra were obtained using a Bruker AC-200 NMR apparatus using the sodium salt of 3-(trimethylsilyl)-propionic acid as internal standard. The following signals were measured (delta  in ppm): 1H-NMR (D2O): delta  2.2 (m, 2H, ---C2---CH2---CO---NH---); delta  2.6 (m, 2H, ---CH2---C2---CO---NH---); delta  2.8---2.9 (m, 2H, ---S---C2---CH---); delta  3.1 (dd, 2H, ---S---C2---CH2---OH); delta  3.2 (dd, 2H, ---S---CH2---C2---OH); delta  3.7-3.9 [m, 3H, HOOC---C(NH2)---CH2--- and HOOC---C2---NH---CO---]; delta  4.6 [m, 1H, ---NH---C(CH2---S---CH2---CH2---OH)CO---]. 13C-NMR (D2O): delta  27.8 (s); delta  33.0 (s); delta  34.3 (s); delta  35.5 (s); delta 46.1 (s); delta  54.8 (p); delta  58.7 (p); delta  61.9 (s); delta  173.6 (q); delta  175.6 (q); delta  176.5 (q); delta  178.0 (q). The signals at delta  54.8 and 58.7 ppm represent the chiral carbon atoms of the gamma -glutamine and cysteine amino acids. The signals at delta  173.6, 175.6, 176.5, and 178 ppm represent the quarternary carbon atoms of the peptide backbone. The signals at delta  27.8, 33.0, 34.3, 35.5, 46.1, and 61.9 ppm represent the carbon atoms of the remaining CH2 groups. Mass spectra were obtained using a Finnigan MAT (Bremen, Germany) MAT-90 with FAB ionization in a matrix of 1-thioglycerol with an Ion Tech 11 NF FAB saddlefield gun (Argon, 6 KeV, 0.4 mA). The following ions (m/z) were observed: 352 ([M + H]+); 374 ([M + Na]+); 396 ([M - H + 2Na]+); 418 ([M - 2H + 3Na]+); 350 ([M - H]-), and 372 ([M - 2H + Na]-). When 2-HEG solutions were made for use in the in vivo experiments, a correction was made for the presence of salts in the final synthetical product.

Dose Excretion Experiments with 1,2-DBE. Rats were individually placed in metabolism cages, and control urine was collected for 24 hr. Next, rats were injected intraperitoneally with a 0.5 ml solution of 1,2-DBE in arachidis oil, or with 0.5 ml arachidis oil alone as a control. The 1,2-DBE doses used were 10, 20, 30, and 40 mg/kg body weight (corresponding to 53, 106, 159, and 212 µmol/kg body weight). Urine, used for the measurement of TDA and 2-HEMA, was collected in two fractions (from 0 to 24 hr and from 24 to 48 hr), after which rats were euthanized by decapitation.

Experiments with CYP2E1 Inhibitors. Rats were placed individually in metabolism cages in the morning. In the afternoon, rats were pretreated orally with 0.25 ml solutions of either DSF, at a dose of 800 mg/kg body weight, or DAS, at a dose of 200 mg/kg body weight, in arachidis oil, or with arachidis oil alone as a control. Rats were subsequently fasted until 1,2-DBE, as a 0.25 ml solution in arachidis oil, at a dose of 40 mg (212 µmol)/kg body weight or CZX, as a 0.25 mol solution in arachidis oil, at a dose of 30 mg (177 µmol)/kg body weight was administered orally the next morning (18 hr after DSF or DAS treatment). Urine, used for the measurement of TDA and 2-HEMA or 6-OH-CZX, was subsequently collected for 24 hr, after which rats were euthanized by decapitation.

Experiments with 2-HEMA and 2-HEG. Rats were placed individually in metabolism cages for 8 hr, after which they were fasted for 18 hr until 2-HEMA or 2-HEG was administered orally as a 0.25 ml solution in 0.9% (w/v) NaCl, at doses of 165 mg (797 µmol)/kg body weight and 215 mg (612 µmol)/kg body weight, respectively. Urine was collected for 24 hr, after which rats were euthanized by decapitation. Alternatively, 2-HEMA or 2-HEG were administered (in the same doses) intravenously as 0.25 ml solutions in 0.9% (w/v) NaCl after the rats had been in the metabolism cages for 1 day. Urine, used for the measurement of TDA and 2-HEMA, was again collected for 24 hr, after which rats were euthanized by decapitation.

Measurement of Urinary TDA and 2-HEMA. Urinary TDA was measured essentially as described by Brakenhof et al. (24). The same method was used to measure 2-HEMA. Urine (1 ml) was acidified by adding 150 µl of 2 M HCl, after which 75 µl of a solution containing the internal standard, BEMA, was added. The BEMA solution was prepared by dissolving 10 mg BEMA in 2.5 ml ethanol, after which 7.5 ml water was added. Acidified fractions were extracted 2 times with 3 ml ethyl acetate, after which the ethyl acetate fractions were combined and evaporated to dryness in a gentle stream of nitrogen. Dry residues were dissolved in 300 µl methanol, after which they were methylated by adding an excess of an ethereal solution of diazomethane for at least 30 min. The solvents and excess diazomethane were evaporated in a gentle stream of nitrogen, and the dry residues were dissolved in 200 µl ethyl acetate before analysis by GC. Calibration curves were made (in rat urine) using commercially obtained TDA and 2-HEMA in the concentration range 10-100 µg/ml and 20-240 µg/ml for TDA and 2-HEMA, respectively. Calibration curves yielded straight lines with the following equations: y = 0.7 + 4.5 * 103 × (r2 = 0.987) and y = 0.6 + 2.3 * 103 × (r2 = 0.991) for TDA and 2-HEMA, respectively. Detection limits were ~5 µg/ml for TDA and 10 µg/ml for 2-HEMA. The GC system used consisted of a Hewlett-Packard 5890 series II gas chromatograph equipped with a FPD using a sulfur selective filter, a 7673A autosampler, and controller and a 3390A integrator. A CPSil5CB column (Chrompack, Middelburg, the Netherlands)---with a length of 25 m, an internal diameter of 0.25 mm, and a stationary phase thickness of 0.2 µm---was used. The oven was programmed from 50°C (1 min) to 288°C, with a rate of 30°C/min. The temperature of the injection port and detector were 250°C. The identity of both TDA and 2-HEMA was confirmed by GC/MS measurements using a Hewlett-Packard 5890 series II gas chromatograph coupled to a Hewlett-Packard 5970 MSD using electron impact ionization (electron energy = 70 eV).

Measurement of Urinary 6-OH-CZX. Urinary 6-OH-CZX was measured essentially as described by Peter et al. (25) and Carrière et al. (26). Urine (10 µl) was acidified by adding 1 ml of 5 M HCl, after which these samples were heated for 30 min at 90°C to hydrolyze glucuronides of 6-OH-CZX. Subsequently, 15 µl of a solution of 2 mg/ml 2-benzoxazolinone (internal standard) was added to each sample. Samples were extracted 2 times with 3 ml ethyl acetate, after which the ethyl acetate layers were combined and evaporated to dryness in a gentle stream of nitrogen. Samples were dissolved in 400 µl of mobile phase (25% methanol/75% water) before analysis by HPLC using UV detection. A calibration curve was made (in rat urine) using commercially obtained 6-OH-CZX in the concentration range of 94-1500 µg/ml. The calibration curve yielded a straight line with the following equation: y = 1.4 * 10-3 + 1.3 * 10-3 × (r2 = 0.994). The detection limit for 6-OH-CZX was ~50 µg/ml. The HPLC system used consisted of a Gilson (Villiers le Bel, France) model 305 pump with a 10WSC pump head, a Gilson model 231 sample injector equipped with a Gilson model 401 dilutor, two Chrompack (Middelburg, the Netherlands) glass columns (100 mm * 3 mm) packed with chromsphere 5C18 (5-µm particle size) reversed-phase material in a cartridge holder also containing a guard column (10 mm * 2 mm), an Applied Biosystems 759A UV detector, and a Hewlett-Packard 3396 series II integrator.

Statistics. Results are represented as means ± SD. The number of animals used per experimental group are indicated in the legends to the figures. Differences between experimental groups were analyzed by the unpaired Student's t test.

    Results
Abstract
Introduction
Results
Discussion
References

Gas chromatograms (GC-FPD) of rat control urine and urine of a rat treated with 1,2-DBE are shown in fig. 2. The identity of both TDA (with a retention time of 4.9 min) and 2-HEMA (with a retention time of 7.4 min), as their methyl esters, was confirmed by the obtained mass spectra (table 1). Furthermore, an as yet unidentified compound was observed with a retention time of 5.9 min. Due to the fact that this peak was obscured by a high degree of background in the GC/MS analysis, no reliable mass spectrum could be obtained for this metabolite.


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  		Fig. 2.   Identification of TDA and 2-HEMA as urinary metabolites of 1,2-DBE.

Gas chromatograms were obtained after extraction of acidified urines obtained from a rat treated with only arachidis oil (A) and a rat treated with 1,2-DBE in arachidis oil (B), as described in Materials and Methods. TDA, with a retention time of 4.9 min, and 2-HEMA, with a retention time of 7.4 min, have been indicated in the chromatogram of urine obtained from a rat treated with 1,2-DBE (B). The internal standard, BEMA, with a retention time of 10.6 min, was added to both samples and is also indicated in the chromatograms.

                              
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TABLE 1
GC/MS data of TDA and 2-HEMA extracted from urine obtained from a rat treated with 1,2-DBE

Both TDA and 2-HEMA were detected as their methyl esters after derivatization of extracted urine samples as described in Materials and Methods.

Dose-excretion curves showed a near linear trend of the urinary excretion of both TDA and 2-HEMA with increasing 1,2-DBE dose (administered intraperitoneally), ranging from 10 to 40 mg/kg body weight (corresponding to 53 to 212 µmol/kg body weight), in urines collected from 0 to 24 hr after administration of 1,2-DBE (fig. 3). Neither of these metabolites were detectable in urines collected from 24 to 48 hr after administration of 1,2-DBE. The percentage of the 1,2-DBE dose excreted as TDA was on average 23.0 ± 3.0% (N = 8), whereas 2-HEMA was excreted at an average of 28.2 ± 8.6% (N = 8) of the 1,2-DBE dose.


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  		Fig. 3.   Dose-excretion relationships for TDA and 2-HEMA.

Dose-excretion relationships are shown for TDA (A) and 2-HEMA (B) excreted in urine after intraperitoneal administration of 1,2-DBE to rats. 1,2-DBE doses used were between 10 and 40 mg/kg body weight (corresponding to 53-212 µmol/kg body weight). Results are the means of two rats for each dose of 1,2-DBE, with error bars representing the SD. Numbers on top of bars are the percentage of the 1,2-DBE dose excreted as TDA or 2-HEMA.

Upon oral administration of 30 mg/kg (177 µmol/kg) CZX, 82.9 ± 26.6% (N = 7) of this compound was excreted as the hydroxylated metabolite, 6-OH-CZX, within 24 hr in the urines of rats. Pretreatment of the rats with either 800 mg/kg DSF or 200 mg/kg DAS, administered orally, led to a significant reduction of the hydroxylation of CZX in both cases. The percentages of CZX hydroxylated to 6-OH-CZX were 15 ± 4.1% (N = 3) (a 81.9% reduction) and 49.3 ± 12.6% (N = 4) (a 40.5% reduction) after pretreatment with DSF or DAS, respectively (fig. 4A). In a similar experimental setup, 1,2-DBE (40 mg/kg, 212 µmol/kg) was administered orally to both control rats and rats pretreated with 800 mg/kg DSF or 200 mg/kg DAS, administered orally. In control rats, 13.1 ± 4.6% (N = 6) of the 1,2-DBE dose was excreted as TDA, while these percentages were 0.4 ± 0.2% (N = 3) (a 97% reduction) and 5.4 ± 4.5% (N = 3) (a 58.8% reduction) in rats pretreated with either DSF or DAS, respectively (fig. 4B). The excretion of 2-HEMA was reduced from 13.8 ± 7.2% (N = 6) in nonpretreated rats to 6.4 ± 1.7% (N = 3) (a 53.6% reduction) after pretreatment with DSF, however, this reduction was not statistically significant (fig. 4C). Pretreatment of rats with DAS did not have any significant effect on the excretion of 2-HEMA, being 16.3 ± 2.4% (N = 3) of the administered 1,2-DBE dose (fig. 4C).


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  		Fig. 4.   Influence of CYP2E1 inhibitors on CYP2E1 activity and 1,2-DBE metabolism.

Excretion of urinary 6-OH-CZX (A), TDA (B), and 2-HEMA (C) are shown after oral administration of 30 mg/kg (177 µmol/kg) CZX in the case of 6-OH-CZX and 40 mg/kg (212 µmol/kg) 1,2-DBE in the case of TDA and 2-HEMA. Results are given for the excretion of these metabolites in control rats and rats pretreated orally with either 800 mg/kg DSF or 200 mg/kg DAS. Results are the means of 7 rats for the CZX group, 3 rats for the CZX + DSF group, 4 rats for the CZX + DAS group, 6 rats for the 1,2-DBE group, and 3 rats for the 1,2-DBE + DSF and 1,2-DBE + DAS groups, with error bars representing the SD. Numbers on Top of bars are the percentages of the administered dose excreted as the indicated metabolites. *p < 0.05; **p < 0.005.

Upon administration of 165 mg/kg (797 µmol/kg) 2-HEMA and 215 mg/kg (612 µmol/kg) 2-HEG, both orally and intravenously to rats, TDA could only be detected [at 1.0 ± 0.4% (N = 3) of the dose] in urines of rats that were treated with 2-HEG orally (table 2). No TDA could be detected in urines of rats that were treated with 2-HEG and 2-HEMA intravenously or with 2-HEMA orally (table 2). To determine whether both 2-HEG and 2-HEMA were absorbed after oral administration, the excretion of 2-HEMA in urine was measured. Upon oral administration of either 2-HEG or 2-HEMA, 29.4 ± 3.2% (N = 3) and 30.4 ± 2.5% (N = 3) of the administered doses were excreted in urine as 2-HEMA, respectively (table 2).

                              
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TABLE 2
Urinary excretion of TDA and 2-HEMA after administration of 2-HEG and 2-HEMA

2-HEG (215 mg/kg; 612 µmol/kg) and 2-HEMA (165 mg/kg; 797 µmol/kg) were administered either orally (solution in arachidis oil) or intravenously [solution in 0.9% (w/v) NaCl] to rats (N = 3 for each compound and administered route), after which TDA and 2-HEMA were measured in urine fractions collected from 0 to 24 hr after administration of the compounds, as described in Materials and Methods.

    Discussion
Abstract
Introduction
Results
Discussion
References

The aim of the present study was to investigate whether TDA is a urinary metabolite of 1,2-DBE and whether or not it can be used as a biomarker selectively reflecting the P450 catalyzed oxidation of 1,2-DBE.

The first approach used to investigate the involvement of P450 in the formation of TDA upon administration of 1,2-DBE was to inhibit selectively the activity of the major P450 isoenzyme involved in the oxidation of 1,2-DBE, being CYP2E1 (16-18). The CYP2E1 inhibitors used in this study were DSF and DAS. DSF has been found to inhibit the enzymatic activity of CYP2E1 toward N-nitrosodimethylamine, in vitro, probably after reduction to DDTC (27). Furthermore, a single dose of DSF reduced CYP2E1 activity toward CZX in vivo in humans (28). In vivo, DSF potentiated both the cytotoxic and carcinogenic effects of 1,2-DBE (29, 30). DAS, one of the constituents of garlic, as well as its metabolites DASO and DASO2, have been shown to inhibit CYP2E1 activity toward N-nitrosodimethylamine, in vitro (31, 32) whereas only DASO2 has been found to inhibit CYP2E1 activity toward p-nitrophenol, in vitro, via a suicide-inhibitory mechanism (33). DAS has also been found to inhibit CYP2E1 activity toward CZX in vivo in rats (34). The inhibitory activity of DAS and its metabolites on CYP2E1 activity might be related to their chemopreventive activity toward the toxicity of some cytotoxic compounds (35-37). The ability of both compounds to inhibit the activity of CYP2E1 in our experimental setup was confirmed by measuring the hydroxylation of CZX to 6-OH-CZX in vivo by measuring 6-OH-CZX excreted in urine. The 6-hydroxylation of CZX is mainly catalyzed by CYP2E1 (25, 38), and this reaction has been used as an in vivo probe for CYP2E1 activity (39, 40). Both DSF and DAS were able to inhibit the CYP2E1 activity by 81.9 and 40.5%, respectively. DSF thus seems to be a stronger CYP2E1 inhibitor when compared with DAS at the doses used in the present study. Other studies support the finding that DSF is a more potent CYP2E1 inhibitor when compared with DAS at doses similar to those used in the present study (27, 32). When these two inhibitors were used in a similar experimental setup in which 1,2-DBE was administered instead of CZX, the excretion of TDA was reduced by 97% and 58.8% using DSF and DAS, respectively. 2-HEMA excretion was reduced after pretreatment with DSF (by 53.6%); however, this reduction was not statistically significant. This points toward a major role of CYP2E1 in the formation of TDA, whereas CYP2E1 contributes to a lesser extent to the formation of 2-HEMA. In a previous study, pretreatment of rats with DSF reduced the excreted amount of 2-HEMA significantly after 1,2-DBE administration (19). However, it was also shown in that study that the effect of DSF pretreatment on the excretion of 2-HEMA was strongly dependent on the 1,2-DBE dose, with the effect of DSF pretreatment being much larger at high 1,2-DBE doses.

In addition to being an inhibitor of CYP2E1 activity, DSF is also known to be an inhibitor of the activity of ALDH, probably after metabolism to S-methyl-N,N-diethylthiocarbamate sulfoxide or the corresponding sulfone (41-44). The role of ALDH in the metabolism of 1,2-DBE has not been thoroughly investigated, however. Because 2-BA, the intermediate product formed by P450, is an aldehyde, the further metabolism of this compound by ALDH might also have been inhibited by DSF. Furthermore, DSF was recently found to inhibit GST activity in vitro by two mechanisms, a reversible inhibition mediated by the reduced form of DSF (i.e. DDTC) and a time-dependent inactivation mediated by DSF itself (45). However, another study has shown that GST activity in vivo was not reduced by DSF (46). In fact, DSF was even shown to be able to induce GST activity upon prolonged exposure in mice (47-49). Therefore, DAS seems to be a more selective inhibitor of CYP2E1, because it does not inhibit either ALDH (50) or GST, which is in fact even induced by DAS (35, 51-53).

As a second approach to study whether TDA is a P450 selective metabolite, intermediate products formed after GST-catalyzed metabolism of 1,2-DBE were administered to rats to determine whether TDA was excreted as a metabolite from these intermediate products. Two compounds were chosen for this purpose: 2-HEMA and 2-HEG. TDA was only detectable in traces after oral administration of 2-HEG; it accounted for only 1.0% of the administered 2-HEG dose. No TDA was detected after intravenous administration of a relatively high dose of 2-HEG, and both oral and intravenous administrations of 2-HEMA. Considering the detection limit for the measurement of TDA, this means that <0.5% of 2-HEG and 2-HEMA was converted to TDA in these cases. In contrast, 2-HEMA was detectable in urine after oral administration of both 2-HEG and 2-HEMA, as 29.4% and 30.4% of the administered doses, respectively. This indicates that both of these compounds were absorbed by the rats after oral administration. Present findings are supported by a previous study in which only 0.5% of an orally administered dose of S-(2-hydroxyethyl)-l-cysteine was excreted as TDA in rats (54).

A proposed schematic representation of the metabolism of reactive intermediates formed after metabolism of 1,2-DBE by either P450 or GST isoenzymes is shown in fig. 5. 2-BA, the reactive intermediate formed upon P450-catalyzed oxidation of 1,2-DBE, is postulated as a precursor of TDA. As was shown in this study, reducing the formation of 2-BA by selective inhibition of the activity of CYP2E1 led to a reduction in the formation of TDA. Furthermore, administration of intermediate products from the GST-catalyzed pathway (i.e. 2-HEG and 2-HEMA) led to excretion of very low to undetectable amounts of TDA. This means that the pathways indicated with an "X" in fig. 5 do not seem to take place in vivo in the rats used in this study.


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  		Fig. 5.   Schematic representation of the metabolism of reactive intermediates, generated from 1,2-DBE, to metabolites that are ultimately excreted in urine.

Formation of GSH conjugates via the P450 route can occur by two pathways. The major pathway will be conjugation of 2-BA to GSH; however, as a minor pathway, 2-BA might first be converted to 2-bromoethanol or 2-bromoacetic acid, after which these compounds conjugate to GSH. This minor pathway is indicated with dashed arrows. As indicated, 2-HEMA is formed from reactive intermediates originating from both P450 and GST-catalyzed metabolism of 1,2-DBE. As shown in the study herein, TDA is only formed from the reactive intermediate 2-BA originating from the P450-catalyzed metabolism of 1,2-DBE. Results also show that the pathways indicated with an "X" (conversion of intermediate products of the GST-catalyzed metabolism of 1,2-DBE to the P450 route) do not take place in vivo in the rat.

In conclusion, it has been shown in this study that TDA is a urinary metabolite of 1,2-DBE, which selectively reflects the P450-catalyzed oxidation of 1,2-DBE in rats. TDA can thus most likely be used as a biomarker of exposure to 1,2-DBE selectively reflecting the amount of 1,2-DBE metabolized by CYP2E1. Together with two already known biomarkers of 1,2-DBE, being 2-HEMA and 2-GEMA, TDA might be useful for biomonitoring purposes, as well as for mechanistic studies toward the metabolism of 1,2-DBE. As shown in the experiments with the CYP2E1 inhibitors, DSF and DAS, the formation of TDA was sensitive toward variability in the activity of CYP2E1 and may also prove to be useful for studies into interindividual variability in biotransformation reactions. Such interindividual variability has recently been reported for the P450-catalyzed metabolism of 1,2-DBE in human liver microsomes (18). Because 1,2-DBE is a carcinogenic compound, studies using these biomarkers might also be used to improve the risk assessment of this compound.

    Acknowledgments

We thank Ed J. Groot for technical assistance with animal experiments and Dr. Ben L. M. van Baar for performing mass spectrometric measurements.

    Footnotes

Received October 30, 1996; accepted January 3, 1997.

   This study was supported by the Dutch Technology Foundation (STW-NWO), Grant VCH22-2831.

Send reprint requests to: Dr. Nico P. E. Vermeulen, Leiden Amsterdam Center for Drug Research, Department of Pharmacochemistry, Division of Molecular Toxicology, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, the Netherlands.

    Abbreviations

Abbreviations used are: 1, 2-DBE, 1,2-dibromoethane; P450, cytochrome P450; GST, glutathione S-transferase; 2-BA, 2-bromoacetaldehyde; GSH, glutathione; 2-HEMA, S-(2-hydroxyethyl)-N-acetyl-l-cysteine (S-(2-hydroxyethyl)mercapturic acid); 2-GEMA, S-[2-(N7-guanyl)ethyl]-N-acetyl-l-cysteine (S-[2-(N7-guanyl)ethyl]mercapturic acid); D4-1, 2-DBE, tetradeutero-1,2-dibromoethane; TDA, thiodiacetic acid; 1, 2-DCE, 1,2-dichloroethane; BEMA, S-benzyl-N-acetyl-l-cysteine (S-benzyl-mercapturic acid); DSF, disulfiram; DAS, diallylsulfide; CZX, chlorzoxazone; 6-OH-CZX, 6-hydroxy-chlorzoxazone; 2-HEG, S-(2-hydroxyethyl)glutathione; CYP, cytochrome P450; FPD, flame photometric detector; MSD, mass selective detector; DDTC, diethyldithiocarbamate; DASO, diallylsulfoxide; DASO2·, diallyl sulfone; ALDH, aldehyde dehydrogenase.

    References
Abstract
Introduction
Results
Discussion
References

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