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butyl ether (ETBE) was studied in rats after inhalation exposure; the biotransformation of the initial metabolite of these ethers, tert-butyl alcohol,...
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Chem. Res. Toxicol. 1998, 11, 651-658

651

Biotransformation of 12C- and 2-13C-Labeled Methyl tert-Butyl Ether, Ethyl tert-Butyl Ether, and tert-Butyl Alcohol in Rats: Identification of Metabolites in Urine by 13C Nuclear Magnetic Resonance and Gas Chromatography/Mass Spectrometry Ulrike Bernauer, Alexander Amberg, Dieter Scheutzow,† and Wolfgang Dekant* Institut fu¨ r Toxikologie, Universita¨ t Wu¨ rzburg, Versbacher Strasse 9, 97078 Wu¨ rzburg, Germany, and Institut fu¨ r Organische Chemie, Universita¨ t Wu¨ rzburg, Am Hubland, 97070 Wu¨ rzburg, Germany Received December 5, 1997

The biotransformation of the fuel oxygenates methyl tert-butyl ether (MTBE) and ethyl tertbutyl ether (ETBE) was studied in rats after inhalation exposure; the biotransformation of the initial metabolite of these ethers, tert-butyl alcohol, was studied after oral gavage. To study ether metabolism, rats were exposed for 6 h to initial concentrations of 2000 ppm of MTBE or ETBE, respectively [2-13C]MTBE and [2-13C]ETBE. Urine was collected for 48 h after the end of the exposure, and urinary metabolites were identified by 13C NMR (13C-labeled ethers) and gas chromatography/mass spectrometry (GC/MS) (12C- and 13C-labeled ethers). To study tert-butyl alcohol metabolism, rats were dosed either with tert-butyl alcohol at natural carbon isotope ratio or with 13C-enriched tert-butyl alcohol (250 mg/kg of body weight), urine was collected, and metabolites were identified by NMR and GC/MS. tert-Butyl alcohol was identified as a minor product of the biotransformation of MTBE and ETBE. In addition, small amounts of a tert-butyl alcohol conjugate, likely a glucuronide, were present in the urine of the treated animals. Moreover, the mass spectra obtained indicate the presence of small amounts of [13C]acetone in the urine of [13C]MTBE and [13C]ETBE-treated rats. 2-Methyl1,2-propanediol, 2-hydroxyisobutyrate, and another unidentified conjugate of tert-butyl alcohol, most probably a sulfate, were major urinary metabolites of MTBE and ETBE as judged by the intensities of the NMR signals. In [13C]-tert-butyl alcohol-dosed rats, [13C]acetone, tert-butyl alcohol, and its glucuronide represented minor metabolites; as with the ethers, 2-methyl-1,2propanediol, 2-hydroxyisobutyrate, and the presumed tert-butyl alcohol sulfate were the major metabolites present. In one human individual given 5 mg/kg [13C]-tert-butyl alcohol orally, 2-methyl-1,2-propanediol and 2-hydroxyisobutyrate were major metabolites in urine detected by 13C NMR analysis. Unconjugated tert-butyl alcohol and tert-butyl alcohol glucuronide were present as minor metabolites, and traces of the presumed tert-butyl alcohol sulfate were also present. Our results suggest that tert-butyl alcohol formed from MTBE and ETBE is intensively metabolized by further oxidation reactions. Studies to elucidate mechanisms of toxicity for these ethers to the kidney need to consider potential toxicities induced by these metabolites.

Introduction The use of oxygen-containing compounds in fuels to reduce harmful engine emissions from cars is required in certain areas of the United States. Chemicals blended with gasoline to meet the required oxygen content of 2.0% (oxygenated gasoline), respectively 2.7% (reformulated gasoline), are referred to as “oxygenates” (1). Methyl tertbutyl ether (MTBE)1 and ethanol are the major oxygenates presently in use; however, other oxygenates such as ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether (TAME) may be increasingly used in the future. Due to possible widespread exposure of humans to these ethers when refueling cars or during commute, several programs * Address correspondence to Dr. W. Dekant. Tel: +49(931)201 3449. Fax: +49(931)201 3446. E-mail: [email protected]. † Institut fu ¨ r Organische Chemie. 1 Abbreviations: MTBE, methyl tert-butyl ether; ETBE, ethyl tertbutyl ether; TAME, tert-amyl methyl ether.

to investigate the toxicology of these compounds are underway (2). The acute toxicity of MTBE and ETBE is low. Both MTBE and tert-butyl alcohol have been studied in longterm bioassays for tumorigenicity. MTBE (inhalation exposure up to 8000 ppm, 6 h/day, 5 days/week for 24 months) and its presumed major metabolite, tert-butyl alcohol (administration in doses up to 5 mg/L in drinking water for 15 months), induce renal tumors in male rats (3, 4). Renal tumor induction by these compounds may be mediated by the accumulation of R2u-globulin (5, 6). An impared degradation of this protein induced by bound metabolites of tert-butyl alcohol and MTBE or by tertbutyl alcohol or MTBE may cause renal toxicity, cell proliferation, and, finally, renal tumors (7). MTBE exposure also increased the incidence of liver tumors in female mice and testicular tumors in male rats (8). Testicular tumors in male rats were also observed

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following oral administration of MTBE in a daily dose of 1000 mg/kg of body weight, 4 days/week for 104 weeks (3). MTBE, ETBE, and their presumed major metabolite tert-butyl alcohol are negative in standard genotoxicity studies (9). As part of the efforts to characterize the toxic effects of MTBE and ETBE in this study, we investigated the biotransformation of MTBE and ETBE. Intensive metabolism of these ethers to tert-butyl alcohol in vivo and further metabolism of tert-butyl alcohol are indicated by a low recovery of tert-butyl alcohol in the urine of humans exposed to MTBE and ETBE under controlled conditions and a high percentage of retention of inhaled MTBE and ETBE likely due to biotransformation to less volatile metabolites (10, 11). The role of biotransformation in the toxic effects of MTBE and ETBE is unknown; studies on the metabolism of these ethers may delineate the role of the parent compound and metabolites in the toxic effects seen. Moreover, the identification of metabolites will help to develop strategies for monitoring exposure of individuals to MTBE and other oxygenates in fuel (12).

Materials and Methods Synthesis of [2-13C]-tert-Butyl Alcohol. An ethereal solution of 60 mmol of CH3J (Merck, Darmstadt, FRG) was slowly added to equimolar amounts of Mg• turnings (1.5 g) covered by 5.0 mL of diethyl ether. The Grignard reaction was initiated by the addition of traces of iodine (15, 16). The mixture was then stirred for 30 min at flux, an ethereal solution of 50 mmol of [2-13C]acetone (CIL Cambridge Isotope Laboratories, Andover, MA, lot no. P-7787) was added within 30 min, and the mixture was kept at flux for 2 h. After cooling, hydrolysis was performed with 50 mL of an ice-cold, saturated NH4Cl solution. The layers were separated, and the aqueous layer was extracted five times with 10 mL of diethyl ether. The ethereal layers were combined and dried over Na2CO3. After evaporation of the solvent the residue was distilled to yield [2-13C]-tert-butyl alcohol (42% yield, 97% GC/FID purity). The structure of the reaction product was confirmed by GC/MS and 1H and 13C NMR. Analytical Characterization of [2-13C]-tert-Butyl Alcohol. 1H NMR (250 MHz, D2O): δ ) 1.25, 9 H [d, JHC ) 4 Hz, (CH3)3COH]. 13C NMR (63 MHz, D2O): δ ) 32.4 [d, JCC ) 39.1 Hz, (CH3)3COH]; 72.6 [s, d, with 0.5% of a satellite doublet, JCC ) 39 Hz, (CH3)3COH]. MS (70 eV): m/z ) 60 [100%, M+ - CH3]; 58 [40%]; 44 [31%]; 42 [40%]. Synthesis of [13C]MTBE and [13C]ETBE from [2-13C]tert-Butyl Alcohol. A mixture of 20 mmol of either methanol (for the synthesis of MTBE) or ethanol (for the synthesis of ETBE) and 3 mL of 10% H2SO4 in H2O was heated to 70 °C; then 10 mmol of [2-13C]-tert-butyl alcohol was added with a syringe. With increasing temperature an azeotropic mixture of MTBE (ETBE), methanol (ethanol), and tert-butyl alcohol was distilled off. Methanol (ethanol) was removed from the reaction mixture by boiling over sodium for 1 h. Further purification was performed by slow distillation with dry ice cooling on a Bu¨chi-GKR-51 Kugelrohr apparatus to yield 26% [2-13C]MTBE and 21% [2-13C]ETBE. The GC/FID purities of both reaction products were >95%. The structures of the reaction products were confirmed by GC/MS and 1H and 13C NMR (17). Analytical Characterization of [2-13C]MTBE. 1H NMR (250 MHz, D2O): δ ) 1.22, 9 H [d, JHC ) 4 Hz, (CH3)3COCH3]; 3.24, 3 H [d, JHC ) 4 Hz, (CH3)3COCH3]. 13C NMR (63 MHz, D2O): δ ) 28.8 [d, JCC ) 40 Hz, (CH3)3COCH3]; 51.6 [s, (CH3)3COCH3]; 77.9 [s, d, with 0.5% of a satellite doublet, JCC ) 40 Hz, (CH3)3COCH3]. MS (70 eV): m/z ) 74 [100%, M+ - CH3]; 58 [59%]; 56 [21%]; 44 [50%]; 42 [38%]. Analytical Characterization of [2-13C]ETBE. 1H NMR (250 MHz, D2O): δ ) 1.15, 3 H [t, J ) 7 Hz, (CH3)3COCH2CH3]; 1.23, 9 H [d, JHC ) 4 Hz, (CH3)3COCH2CH3]; 3.54, 2 H [q, J )

Bernauer et al. 7 Hz, JHC ) 2 Hz, (CH3)3COCH2CH3]. 13C NMR (63 MHz, D2O): δ ) 17.9 [s, (CH3)3COCH2CH3]; 29.3 [d, JCC ) 39 Hz, (CH3)3COCH2CH3]; 60.2 [s, (CH3)3COCH2CH3]; 77.7 [s, d, with 0.5% of a satellite doublet, JCC ) 40 Hz, (CH3)3COCH2CH3]. MS (70 eV): m/z ) 88 [94%, M+ - CH3]; 60 [100%]; 58 [88%]; 56 [12%]; 44 [23%]; 42 [45%]; 41 [16%]. Synthesis of 2-Methyl-1,2-propanediol. 2-Methyl-1,2propanediol was prepared by reduction of the ethyl ester of 2-hydroxyisobutyrate; 4 mmol of NaBH4 (151 mg) was dissolved in 12 mL of 2-propanol followed by addition of 10 mmol of 2-hydroxybutyric acid ethyl ester at room temperature. The mixture was then stirred overnight. To dissolve the precipitated solid 2 N HCl was added to the reaction mixture. After extraction with ethyl ether the organic layers were combined and dried over K2CO3. After removal of the solvent, 2-methyl1,2-propanediol was isolated by fractional distillation under reduced pressure. Analytical Characterization of 2-Methyl-1,2-propanediol. 1H NMR (250 MHz, D O): δ ) 1.19, 6 H [s, (CH ) C(OH)CH 2 3 2 2 (OH)]; 3.42, 2 H [s, (CH3)2C(OH)CH2(OH)]. 13C NMR (63 MHz, D2O): δ ) 27.4 [s, (CH3)2C(OH)CH2(OH)]; 72.6 [s, (CH3)2C(OH)CH2(OH)]; 74.2 [s, (CH3)2C(OH)CH2(OH)]. MS (70 eV): m/z ) 75 [8%, M+ - CH3]; 59 [70%]; 57 [28%]; 55 [12%]; 43 [100%]; 42 [22%]; 41 [33%]. Animals and Treatment. Male and female Fischer F344 rats (220-260 g) were obtained from Harlan Winkelmann (Borchen, FRG). Animals were kept at constant humidity and temperature (21 °C) in the animal facility of the department with a 12-h light/dark cycle in steel cages (4 rats/cage). Food (Altromin) and tap water were provided ad libitum. Before the experiments, the animals were accustomed to the metabolic cages for 3 days; control urine was collected during this time for 12 h before the exposure. Experimental Design. Male and female rats were individually exposed to [12C]- or [13C]MTBE (separate experiments with two male and two female rats for each [12C]- and [13C]MTBE and [12C]- and [13C]ETBE) by inhalation for 6 h in a static exposure chamber; initial concentrations of MTBE or ETBE were 2000 ppm. After the end of the 6-h exposures, the animals were individually housed in all glass metabolic cages with free access to food (Altromin) and tap water in the animal facility of the department. Urine samples were collected in 24-h intervals for 48 h and analyzed by 13C NMR (experiments with 13C-labeled ethers) and GC/MS. Male rats (n ) 3/experiment) were treated with either [12C]- or [13C]-tert-butyl alcohol by gavage (250 mg/ kg in corn oil). All animals were individually kept in metabolic cages for 72 h. After the termination of the experiments, all animals were sacrificed by cervical dislocation. Exposure of One Human Volunteer to [13C]-tert-Butyl Alcohol. [13C]-tert-Butyl alcohol was orally administered in a gel capsule at a dose of 5 mg/kg to one human volunteer (male, age 44 years, body weight 80 kg). Urine was collected in 12-h intervals for 48 h and analyzed by 13C NMR as described below. The human experiment was approved by the institutional review board of the University of Wu¨rzburg. Chamber Design and Treatment. The design of the exposure chamber has been described in detail (13). Volume of the system built from stainless steel was 1.89 L. The chamber atmosphere was circulated (2 L/min) by a stainless steel-bellows pump. Carbon dioxide was removed with barium hydroxide contained in a scrubber on the output side of the chamber downstream from a moisture condenser and a flow meter. Chamber oxygen concentrations were maintained within the range of 20-22% by an oxygen monitor which was connected to a solenoid valve that metered oxygen into the chamber as required. The animals were introduced into the chamber at 10 a.m., and the calculated amounts of the 12C- or 13C-labeled ethers were introduced with a microliter syringe. The concentrations of the ethers in the gas phase of the chamber were monitored every 10 min by an automatic gas sampling valve; 100 µL of the gas phase was introduced into a capillary gas chromatograph (HP 5970) and separated with a 40-m DB1-coated fused silica

Biotransformation of Fuel Oxygenates

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Scheme 1. Synthetic Routes to [13C]MTBE and [13C]ETBE from [13C]Acetone

column (DB1, J&W Scientific; 40 m, i.d. ) 0.18 mm, film thickness ) 0.4 µm) at an oven temperature of 30 °C using hydrogen as carrier gas. Ether concentrations in the air were quantified by flame ionization detection (FID). Instrumental Analysis and Sample Processing. Before the recording of 13C NMR spectra, the urines obtained from the animals exposed to 13C-labeled ethers and control urine (720 µL) were introduced into a NMR tube (5-mm i.d.) and 80 µL of D2O was added. These samples were directly analyzed by NMR. Some of the urine samples were treated with β-glucuronidase (Sigma, G7846, lot no. 72H6836) for 30 min at 37 °C or sulfatase (Sigma, S9751, lot no. 621-07881) for 48 h at 37 °C (14); aliquots of the treated urine samples were then also analyzed by NMR. For nonenzymatic cleavage urine samples were acidified to pH 2 with concentrated hydrochloric acid and incubated for 1 h at 37 °C. For gas chromatography/mass spectrometry, 0.5 mL of the urine samples was introduced into 1.5-mL sample vials, acidified with 2 N HCl to pH 4, and kept at 80 °C for 30 min; 100 µL of the headspace from the incubations was then injected into the GC. To analyze less volatile metabolites, some samples (0.5 mL) were extracted with 0.5 mL of ethyl acetate and 1 µL of the ethyl acetate layer was analyzed via GC/MS. Separation was performed on a 30-m × 0.25-mm fused silica column coated with DB-WAX, film thickness 0.25 µm. For analysis, a temperature gradient from 35 to 230 °C with a heating rate of 10 °C/min was applied. Electron impact mass spectra (70 eV) were recorded and metabolite peaks identified by comparison of the chromatograms of the urine from treated rats with those of untreated controls. 1H NMR and composite pulse-decoupled 13C NMR spectra were recorded with a Bruker AC 250 spectrometer or a Bruker Avance DMX 600 NMR spectrometer under standard conditions. Usually, 2000 scans were aquired for Fourier transformation. Gas chromatography/mass spectrometry was performed with a Fisons MD 800 mass spectrometer coupled to a Carlo Erba GC 8000 series gas chromatograph. Split injection (split ratio 1:5) was used, and spectra were recorded from m/z 30 to 300 in 1-s intervals.

Results 13C-Labeled

Synthesis of Ethers. To identify metabolites of the ethers without the expensive synthesis of 14C-labeled material and to be able to detect nonvolatile metabolites, 13C-labeled ethers (>99% 13C in one carbon atom) were prepared (Scheme 1). The use of stable isotope-labeled ethers permits metabolite analysis in urine by 13C NMR in sufficient sensitivity, also permits the identification of metabolites not anticipated, and confirms the structure of metabolites. The 13C-labeled ethers were synthesized according to standard procedures; the purity of all compounds used for animal exposures was >95%. The major impurity present was [2-13C]-tert-butyl alcohol. The presence of this compound

does not interfere with the intended identification of metabolites by NMR spectrometry since the alcohol is also formed as a metabolite from the ethers in vivo. Moreover, the synthetic procedures applied also yielded [2-13C]-tert-butyl alcohol which could be also used for metabolism studies for a better definition of the fate of this first intermediary metabolite of MTBE and ETBE in vivo in rats and humans. Monitoring of the exposure chamber air concentrations in MTBE and ETBE exposures of rats indicated a continuous decrease of the air concentrations of the compounds due to uptake of the ethers by the rats and metabolism to less volatile metabolites. At the end of the exposure after 6 h the ether concentrations in the chamber were below 100 ppm indicating intensive metabolism of the ethers by the rats. Analysis of the gas phase of the chamber did not indicate the formation of volatile or exhaled ether metabolites detectable by flame ionization detection. Urine samples were collected in 24-h intervals for 48 h and analyzed by GC/MS and 13C NMR. A typical NMR spectrum of a urine sample is shown in Figure 1. Biotransformation of MTBE. The NMR spectra obtained from the urine of [13C]MTBE-exposed rats showed several resonances which were also present in the urine from control animals (not shown in Figure 1). The structures of those endogenous products were assigned by comparison with literature data and reference spectra (18, 19). In addition, the range of chemical shifts between 70 and 90 ppm, where only one resonance was observed in the 13C NMR spectra of control urine (δ ) 78.3), showed several signals indicative of MTBE metabolites (δ ) 73.7, 74.3, 76.6, 80.5). The structures of these metabolites were elucidated by a combination of 13 C NMR and GC/MS. The NMR signal at δ ) 73.7 most likely represents a glucuronide of tert-butyl alcohol since this signal disappeared after treatment of urine with glucuronidase with concomitant appearance of the signal for tert-butyl alcohol at δ ) 72.6. Moreover, the signal also disappeared and the tert-butyl alcohol signal appeared when the urines were treated with acid to cleave acid-labile conjugates such as glucuronides. Calculations to predict the structure based on the chemical shift of the signal at δ ) 74.2 suggest that this metabolite may represent 2-methyl-1,2-propanediol. The 13 C NMR spectrum of synthetic 2-methyl-1,2-propanediol also showed a resonance at δ ) 74.2 attributed to the C2 atom. The presence of 2-methyl-1,2-propanediol is also suggested by the results of mass spectrometric analysis of urine from MTBE-treated rats (Figure 2). The mass

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Figure 3. Mass spectra of [13C]acetone present in the urine of a male Fischer 344 rat exposed to [13C]MTBE (spectrum B). The major fragments are shifted by 1 mass unit indicating the presence of 13C in these fragments.

Figure 1. (A) 63-MHz 13C NMR spectrum of urine from an untreated female Fischer 344 rat serving as control; (B) 63-MHz 13C NMR spectrum of a 24-h urine sample from a female rat exposed to 2000 ppm [13C]MTBE for 6 h; 2000 scans were recorded for Fourier transformation. Only the shift range between 70 and 90 ppm is shown. The following structural assignments were made: δ ) 80.5, tert-butyl alcohol sulfate; δ ) 76.6, 2-hydroxyisobutyrate; δ ) 74.3, 2-methyl-1,2-propanediol; δ ) 73.7, tert-butyl alcohol glucuronide.

Figure 2. Mass spectrum of 2-methyl-1,2-propanediol present in the urine (collected for 24 h after the end of exposure) of a male Fischer 344 rat exposed to [12C]MTBE (2000 ppm) for 6 h. A compound with a similar mass spectrum with several fragments shifted by 1 mass unit was observed in the urine of rats exposed to [13C]MTBE.

spectrum of the [12C]MTBE metabolite in urine and that of the synthetic reference were identical confirming 2-methyl-1,2-propanediol as a urinary metabolite of MTBE. Moreover, in the mass spectrum of the peak representing 2-methyl-1,2-propanediol after separation of the urine of rats treated with [13C]MTBE, several signals were shifted by 1 mass unit, further supporting the assigned structure (m/z ) 76, 60, 58, 56, 44, 42, 40). The NMR signal at δ ) 76.6 exhibited an identical chemical shift as the C2 atom of hydroxyisobutyrate and

was greatly increased when authentic hydroxyisobutyrate was added to the urine sample. This observation confirms hydroxyisobutyrate as a metabolite of [13C]MTBE. The structure of 2-hydroxyisobutyrate was also confirmed by mass spectrometry (data not shown). The NMR signal at δ ) 80.6 which also represents a major metabolite of MTBE in rat urine was not conclusively identified. The chemical shift of the signal indicates the presence of an electron-withdrawing group next to the C atom carrying the 13C label but no major structural change in the molecule. The NMR signal was decreased in intensity by 80% (relative to the urea signal at δ ) 165.5) when urine samples were treated for a prolonged time (48 h at 37 °C) with sulfatase with a parallel increase in the intensity of the signal representing tert-butyl alcohol. Moreover, the signal disappeared and a tert-butyl alcohol resonance appeared when urine samples were treated with acid to cleave acid-labile conjugates such as sulfates. In summary, these data suggest that the metabolite represents a conjugate of tertbutyl alcohol, likely a sulfate. Comparison of the NMR spectra recorded from the urine of the treated male and female rats did not indicate relevant differences between the individual animals and also did not indicate sex differences in the structures of metabolites formed or in relative signal intensities when the spectra were recorded under identical acquisition conditions. Identical metabolites were also present, albeit at lower concentrations, in the urines of both male and female rats treated with [13C]MTBE collected between 24 and 48 h after exposure. In addition to structure elucidation of metabolites by 13C NMR, all urine samples obtained in these studies were also analyzed by headspace GC/MS. In addition to the metabolites suggested by NMR analysis in the urines of rats treated with [13C]MTBE, the peak representing tert-butyl alcohol was shifted by 1 mass unit and the peak representing acetone showed fragments representing both [12C]acetone and [13C]acetone (Figure 3) indicating the formation of [13C]acetone as a metabolite of [13C]MTBE. The concentrations of [13C]acetone present were, however, too low to result in a signal in the 13C NMR spectra. Biotransformation of ETBE. A typical NMR spectrum of a urine sample from [13C]ETBE-exposed rats is shown in Figure 4. The chemical shifts of the observed signals were identical to the signals observed in the urines of animals exposed to [13C]MTBE. Therefore, the metabolites formed are identical with those resulting from MTBE metabolism (δ ) 73.7, tert-butyl alcohol

Biotransformation of Fuel Oxygenates

Figure 4. 63-MHz 13C NMR spectrum of a 24-h urine sample from a female Fischer 344 rat exposed to 2000 ppm [13C]ETBE for 6 h; 2000 scans were recorded for Fourier transformation. Only the shift range between 70 and 90 ppm is shown. The following structural assignments were made: δ ) 80.5, tertbutyl alcohol sulfate; δ ) 76.6, 2-hydroxyisobutyrate; δ ) 74.3, 2-methyl-1,2-propanediol; 73.7, tert-butyl alcohol glucuronide.

glucuronide; δ ) 74.3, 2-methyl-1,2-propanediol; δ ) 76.6, 2-hydroxyisobutyrate; δ ) 80.5, tert-butyl alcohol sulfate). This indicates that the major ETBE metabolites, as well as the major MTBE metabolites, result from tert-butyl alcohol formed in the first metabolic step of the ether biotransformation. Again, no significant differences in signal intensities were observed in the urine of male and female rats exposed to [13C]ETBE. In the urine collected between 24 and 48 h after exposure, only the signal at δ ) 76.6 ppm was present, indicating that 2-hydroxyisobutyrate is excreted slowly and over a relatively long period. Neither high-resolution NMR nor GC/MS analysis of the urine samples from ETBE-exposed rats indicated the presence of ETBE metabolites formed by oxidation of the β-carbon of the ethyl group in ETBE (e.g., tert-butyl glycol or tert-butoxyacetic acid). Small amounts of [13C]acetone were also present in the urines of rats exposed to [13C]ETBE as indicated by GC/MS of the headspace from urine samples. Biotransformation of tert-Butyl Alcohol. Studies on the metabolism of [13C]-tert-butyl alcohol were included to confirm the structures of metabolites “downstream” from the formation of tert-butyl alcohol and to identify if metabolic reactions of MTBE at sites other than the methyl ether moiety may occur. Moreover, [13C]tert-butyl alcohol was available in sufficient amounts from the synthesis of the 13C-labeled ethers. A typical NMR spectrum of a urine sample from tert-butyl alcoholtreated rats is shown in Figure 5. Again, at chemical shifts between 70 and 90 ppm, where only one resonance (δ ) 78.5) of an endogenous compound was present, several signals indicative of tertbutyl alcohol metabolites (δ ) 72.6, 73.7, 74.3, 76.6, 80.4) were observed. In addition, several minor signals representing either minor metabolites (not observed in other experiments due to lower concentrations of 13C in urine) or coupling of the 13C atom with 12C atoms were observed. The signal at δ ) 72.6 represents tert-butyl alcohol. This compound was identified by comparison of the δ-value with that of a synthetic reference. Moreover, addition of [13C]-tert-butyl alcohol greatly increased the intensity of this signal. Analysis of the headspace of urine samples from [12C]- and [13C]MTBE-treated rats by GC/MS also confirmed the presence of [12C]- and [2-13C]tert-butyl alcohol. The chemical shifts of the other signals

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Figure 5. 63-MHz 13C NMR spectrum of a 24-h urine sample from a male Fischer 344 rat administered [13C]-tert-butyl alcohol (250 mg/kg) by gavage; 2000 scans were recorded for Fourier transformation. The following structural assignments were made: δ ) 80.4, tert-butyl alcohol sulfate; δ ) 76.6, 2-hydroxyisobutyrate; δ ) 74.3, 2-methyl-1,2-propanediol; δ ) 73.7, tertbutyl alcohol glucuronide; δ ) 72.6, tert-butyl alcohol.

Figure 6. 63-MHz 13C NMR of a 24-h urine sample from a male volunteer administered 5 mg/kg [13C]-tert-butyl alcohol. The following structural assignments were made: δ ) 76.6, 2-hydroxyisobutyrate; δ ) 74.3, 2-methyl-1,2-propanediol; δ ) 73.7, tert-butyl alcohol glucuronide; δ ) 72.6, tert-butyl alcohol; δ ) 72.2 unknown.

were identical to those seen in urine after [13C]MTBE inhalation exposure. On the basis of these observations and MS data identical to those obtained with MTBE, tertbutyl alcohol, tert-butyl alcohol glucuronide, and tertbutyl alcohol sulfate, 2-hydroxyisobutyrate and 2-methyl1,2-propanediol are identified as urinary metabolites of tert-butyl alcohol. Identical metabolites were also present, albeit at lower concentrations, in the urines collected between 24 and 48 h after exposure. In contrast to the MTBE inhalation exposure, where 2-hydroxyisobutyrate was the most prominent metabolite based on signal intensities after acquiring spectral data under identical conditions, after tert-butyl alcohol exposure, the presumed tert-butyl alcohol sulfate was the main metabolite excreted. Headspace analysis by GC/MS also indicated the presence of small amounts of [13C]acetone in the urines of [13C]-tertbutyl alcohol-treated animals. In the urine of the human individual administered [13C]-tert-butyl alcohol orally, the 13C NMR spectra recorded (Figure 6) showed the presence of tert-butyl alcohol, tert-butyl alcohol glucuronide, 2-hydroxyisobutyrate and 2-methyl-1,2 propanediol. Moreover, an additional NMR resonance (δ ) 72.2) not observed in control urine taken before or 1 week after tert-butyl alcohol

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Scheme 2. Biotransformation of MTBE/ETBE and tert-Butyl Alcohol (TBA) in Ratsa

a

Excreted metabolites in urine are underlined.

administration was present in the urine samples. The structures of this metabolite could not be elucidated. The metabolites were present in all urine samples analyzed; in contrast to rat urine samples where the presumed tertbutyl alcohol sulfate was observed as a major product excreted after tert-butyl alcohol administration, the sulfate was only present in traces in the human urine samples analyzed. In the human urine, 2-hydroxyisobutyrate was the major metabolite excreted based on relative intensities of the 13C NMR signals.

Discussion The biotransformation of MTBE, ETBE, and their presumed major metabolite tert-butyl alcohol was studied in rats. In addition, the biotransformation of tert-butyl alcohol was studied in one human individual. The major aim of the study was to identify metabolites formed and excreted in urine. The use of 13C-labeled ethers permitted detection and identification of metabolites by 13C NMR. Moreover, comparison of the mass spectra of urine constituents from animals treated with 12C- or 13C-labeled compounds also permitted identification of peaks representing metabolites due to the difference of 1 mass unit in selected fragments of 13C-containing metabolites and identification of metabolites of the [13C]ethers which were also formed endogenously (e.g., acetone). The MTBE studies confirmed tert-butyl alcohol, 2-methyl-1,2-propanediol, and 2-hydroxyisobutyrate as MTBE metabolites excreted in urine (20). Metabolite excretion was not quantified in this study; however, other studies have demonstrated that biotransformation significantly contributes to the disposition of MTBE in rats. For example, 65% of the radioactivity was recovered in the

urine of rats within 48 h after exposure to 400 ppm [14C]MTBE for 6 h indicating intensive metabolism (21). On the basis of the structures of the metabolites elucidated, MTBE biotransformation in the rat proceeds by oxidation of the methyl group in MTBE to give an intermediate hemiacetal which decomposes to tert-butyl alcohol with release of formaldehyde (Scheme 2). tert-Butyl alcohol is also formed in vitro from MTBE by a cytochrome P450-mediated oxidation reaction (22, 23). The involvement of cytochromes P450 2E1 and P450 2A6 in the transformation of MTBE to tert-butyl alcohol in human liver microsomes has been established. In [13C]ETBE-exposed rats, identical metabolites were identified as observed in MTBE-treated rats. On the basis of relative signal intensities after identical acquisition conditions, the concentrations of metabolites present in the urines of [13C]ETBE-treated animals within 48 h after the end of the exposures were similar to those observed in the urine of [13C]MTBE animals exposed under identical conditions. This observation indicates that there is a similar extent of metabolism of both ethers to metabolites excreted in urine of rats after inhalation exposure. These metabolite structures also suggest that tert-butyl alcohol is also a major intermediate metabolite of ETBE formed by a cytochrome P450-mediated oxidation reaction. With ETBE, oxidation by cytochrome P450 seems to occur exclusively at the R-carbon atom of the ethyl ether moiety since metabolites whose formation could be explained by oxidation of the β-carbon (such as tert-butyl glycol) were not detected. The finding of a preferred oxidation on the R-carbon is in line with observations on the biotransformation of ethyl ether where ether cleavage by R-carbon oxidation is the major

Biotransformation of Fuel Oxygenates

pathway of biotransformation (24). Moreover, aliphatic hydrocarbons such as n-hexane are also preferentially oxidized at the w - 1 atom (25). In ETBE biotransformation, the intermediate hemiacetal also decomposes to give tert-butyl alcohol and acetaldehyde. The further fate of the aldehydes formed in MTBE and ETBE biotransformation has not been investigated; they are, however, expected to be rapidly metabolized to formate and acetic acid. The low amounts of tert-butyl alcohol recovered as the free alcohol in urine of MTBE- and ETBE-exposed rats and the presence of tert-butyl alcohol conjugates and further metabolites downstream from tert-butyl alcohol indicate intensive further metabolism of tert-butyl alcohol in rats. Intensive metabolism of tert-butyl alcohol likely formed as an intermediate in human MTBE biotransformation is also indicated by the low recovery of tertbutyl alcohol in the urines of human volunteers exposed to MTBE (10). Intensive biotransformation of tert-butyl alcohol in rodents both by conjugation and by oxidation is confirmed by results of our experiments with administration of [13C]-tert-butyl alcohol. The NMR spectra obtained from the urines of [13C]-tert-butyl alcohol-treated rats and the human individual exposed to [13C]-tert-butyl alcohol showed identical metabolites (tert-butyl alcohol conjugates, 2-methyl-1,2-propanediol, and 2-hydroxyisobutyrate) as observed in the urines of MTBE- and ETBEtreated rats. Conjugation of tert-butyl alcohol with activated glucuronic acid results in the excretion of the glucuronide conjugate. A glucuronide conjugate of tert-butyl alcohol has been described in rabbits (26), and these authors recovered approximately 25% of the dose of tert-butyl alcohol as glucuronide. Our study also provides indirect evidence for the formation of a sulfate conjugate of tertbutyl alcohol which, based on the intensities of the 13C signal in the NMR spectra of the urine from animals treated with 13C-labeled ethers and [13C]-tert-butyl alcohol, represents a major pathway of tert-butyl alcohol biotransformation in the rat. The low recovery of the presumed tert-butyl alcohol sulfate in the urine of the human individual exposed to [13C]-tert-butyl alcohol is likely based on a low affinity of human sulfotransferase(s) for tert-butyl alcohol. Species differences in the capacities and substrate specificities of sulfotransferases have been described (27). The two other metabolites present in the urine of tert-butyl alcohol-treated animals and also in urine of the rats exposed to MTBE and ETBE (2-methyl-1,2-propanediol and 2-hydroxyisobutyrate) suggest further oxidative metabolism of tert-butyl alcohol. The likely pathway for the formation of these metabolites involves oxidation of tert-butyl alcohol by cytochromes P450 to give 2-methyl-1,2-propanediol. tert-Butyl alcohol is not a substrate for alcohol dehydrogenase but is oxidized by rat liver microsomes to formaldehyde and acetone under conditions consistent with an involvement of cytochromes P450 (28, 29). Cytochrome P450-mediated oxidation of a C-H bond in one of the methyl groups of tert-butyl alcohol results in the excretion of the diol metabolite. Further oxidation of 2-methyl-1,2-propanediol results in 2-hydroxyisobutyrate which is also excreted as a major metabolite of tert-butyl alcohol, MTBE, and ETBE. In this study, tert-butyl alcohol metabolism to small amounts of acetone in vivo was also observed. [13C]-tert-

Chem. Res. Toxicol., Vol. 11, No. 6, 1998 657

Butyl alcohol as the origin of the excreted [13C]acetone is confirmed by the obtained mass spectra confirming the presence of 13C. Acetone is likely formed by further oxidation of 2-hydroxyisobutyrate. Acetone was recovered only as a very minor metabolite in the urine; other studies on the biotransformation of tert-butyl alcohol in rodents revealed inconsistent results in regard to acetone formation: some authors reported significant increases in blood acetone concentrations after tert-butyl alcohol administration, while others failed to detect acetone as a product formed in vivo from tert-butyl alcohol (28, 30). Based on the known capacity of the human liver to oxidize MTBE and ETBE to tert-butyl alcohol and formaldehyde (23), and the results on the metabolism of tert-butyl alcohol in one human, it may be anticipated that there is a qualitatively identical biotransformation of MTBE and ETBE in rats and humans. Presently, the contribution of biotransformation reactions and of metabolites formed to the toxic effects observed after repeated administration of MTBE, ETBE, and tert-butyl alcohol is unclear. Both MTBE and tert-butyl alcohol induce nephropathy in rats and were found to increase the incidence of renal tumors in male rats (3, 4, 31). The R2u-globulin nephropathy syndrome has been suggested to be involved in formation of renal tumors in male rats after MTBE and tert-butyl alcohol. R2u-Globulin nephropathy involves an impaired degradation of the male ratspecific protein R2u-globulin in the kidney induced by xenobiotics bound to this protein. The impaired degradation in the lysosomes of the kidney results in accumulation of the modified R2u-globulin in the kidney tubular epithelial cells, lysosomal rupture, cell death, and cell proliferation. The induced cell proliferation is suggested to be a major contributor to tumor formation. Tumors are only observed in male rats due to the sex and speciesspecific biosynthesis of R2u-globulin (7). The chemical responsible for a possible binding to R2u-globulin in MTBE and tert-butyl alcohol effects is not defined (4, 32). On the basis of in vitro studies, unchanged MTBE may be responsible for binding (33); however, a role for tert-butyl alcohol formed as a metabolite may not be ruled out. However, MTBE is only a weak inducer of R2u-globulin accumulation, and other mechanisms may contribute to toxic effects of this ether to the kidney. Our results demonstrate that other metabolites than tert-butyl alcohol are also formed in vivo from MTBE and ETBE and effects induced by these metabolites may also contribute to the toxicities observed. The MTBE metabolites observed in this study in rodents after inhalation of high concentrations of MTBE (necessary for obtaining sufficient amounts of metabolites for conclusive identification) are also excreted in humans exposed to low and occupationally relevant concentrations of MTBE (40 ppm) (Amberg et al., unpublished).

Acknowledgment. Research desribed in this article was conducted under contract from the Health Effects Institute (HEI, Research Agreement No. 96-3), an organization jointly funded by the United States Environmental Protection Agency (EPA) (Assistance Agreement X-816285) and certain motor vehicle and engine manufacturers. The contents of this article do not necessarily reflect the views of HEI, or its sponsors, nor do they necessarily reflect the views and policies of EPA or motor vehicle and engine manufacturers. Parts of this work were also supported by the Biomed Program of the

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Bernauer et al.

European Union, Contract No. BMH4-CT96-0184.

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