Biotransformation and Kinetics of Excretion of Ethyl tert-Butyl Ether in Rats and Humans

Alexander Amberg, Elisabeth Rosner and Wolfgang Dekant1

Institut für Toxikologie, Universität Würzburg, Versbacher Strasse 9, 97078 Würzburg, Germany

Received July 6, 1999; accepted September 29, 1999


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ethyl tert-butyl ether (ETBE) may be used in the future as an additive to gasoline to increase oxygen content and reduce tailpipe emissions of pollutants. Therefore, widespread human exposure may occur. To contribute to the characterization of potential adverse effects of ETBE, its biotransformation was compared in humans and rats after inhalation exposure. Human volunteers (3 males and 3 females) and rats (5 males and 5 females) were exposed to 4 (4.5 ± 0.6) and 40 (40.6 ± 3.0) ppm ETBE for 4 h in a dynamic exposure system. Urine samples from rats and humans were collected for 72 h at 6-h intervals, and blood samples were taken in regular intervals for 48 h. In urine, ETBE and the ETBE-metabolites tert-butanol (t-butanol), 2-methyl-1,2-propane diol, and 2-hydroxyisobutyrate were quantified; ETBE and t-butanol were determined in blood samples. After the end of the exposure period to inhalation of 40-ppm ETBE, blood concentrations of ETBE were found at 5.3 ± 1.2 µM in rats and 12.1 ± 4.0 µM in humans. The ETBE blood concentrations, after inhalation of 4-ppm ETBE, were 1.0 ± 0.7 µM in rats and 1.3 ± 0.7 µM in humans. ETBE was rapidly cleared from blood. After the end of the 40-ppm ETBE exposure period, the blood concentrations of t-butanol were 13.9 ± 2.2 µM in humans and 21.7 ± 4.9 µM in rats. After 4-ppm ETBE exposure, blood concentrations of t-butanol were 1.8 ± 0.2 µM in humans and 5.7 ± 0.8 µM in rats. t-Butanol was cleared from human blood with a half-life of 9.8 ± 1.4 h in humans after 40-ppm ETBE exposure. In urine samples from controls and in samples collected from the volunteers and rats before the exposure, low concentrations of t-butanol, 2-methyl-1,2-propane diol, and 2-hydroxyisobutyrate were present. In the urine of both humans and rats exposed to ETBE, the concentrations of these compounds were significantly increased. 2-Hydroxyisobutyrate was recovered in urine as the major excretory product formed from ETBE; t-butanol and 2-methyl-1,2-propane diol were minor metabolites. All metabolites of ETBE excreted with urine were rapidly eliminated in both species after the end of the ETBE exposure. Excretion half-lives for the different urinary metabolites of ETBE were between 10.2 and 28.3 h in humans and 2.6 and 4.7 h in rats. The obtained data indicate that ETBE biotransformation and excretion are similar for rats and humans, and that ETBE and its metabolites are rapidly excreted by both species. Between 41 and 53% of the ETBE retained after the end of the exposure was recovered as metabolites in the urine of both humans and rats.

Key Words: ethyl tert-butyl ether (ETBE); tert-butanol (t-butanol); ETBE inhalation exposure; ETBE as gasoline additive; ETBE biotransformation and excretion half-lives; rat; human..


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The presence of oxygen-containing compounds to reduce harmful engine emissions from automobiles is required in certain areas of the United States. Chemicals blended with gasoline hydrocarbons to meet the required oxygen content of 2.0% for oxygenated gasoline or 2.7% for reformulated gasoline are referred to as "oxygenates" (Costantini, 1993Go). Methyl tert-butyl ether (MTBE) and ethanol are the major oxygenates presently in use. However, compounds such as ethyl tert-butyl ether (ETBE) or tert-amyl-methyl ether (TAME) may be used in the future. Due to possible widespread exposure of humans to these ethers when refueling cars or while commuting, several programs to study the toxicology of these compounds are under way (White et al., 1995Go).

The acute toxicity of ETBE is low, and other data on ETBE toxicity are scarce. However, the structurally related MTBE and tert-butanol (t-butanol), a metabolite of both MTBE and ETBE, have been studied in long-term bioassays for tumorigenicity (for review, see (Bird et al., 1997Go; ECETOC, 1997Go; Rudo, 1995Go). MTBE and t-butanol induce renal tumors in male rats (Burleigh-Flayer et al., 1992Go; Takahashi et al., 1993Go). Renal tumor induction by these compounds may be mediated by the accumulation of {alpha}2u-globulin (Borghoff et al., 1996Go; Scientific Advisory Board on Toxic Air Pollutants, 1995Go). An impaired degradation of this protein induced by bound metabolites of t-butanol and MTBE or by t-butanol or MTBE may cause renal toxicity, cell proliferation, and finally, renal tumors (Swenberg et al., 1989Go). ETBE and its metabolite t-butanol are negative in standard genotoxicity studies (Duffy et al., 1992Go).

As a part of the effort to characterize the toxic effects of ETBE, we investigated the biotransformation and toxicokinetics of ETBE in rats and in human volunteers. Intensive metabolism of ETBE to t-butanol and further metabolism of t-butanol were indicated by a low recovery of t-butanol in the urine of humans exposed to ETBE and a high percentage of retention of inhaled ETBE (Nihlen et al., 1998aGo). In rats and humans exposed to ETBE by inhalation, t-butanol, 2-methyl 1,2-propane diol and 2-hydroxyisobutyrate were excreted as metabolites in urine (Fig. 1Go). Moreover, 2-methyl 1,2-propane diol and 2-hydroxyisobutyrate were also identified as metabolites formed from t-butanol in rodents and humans (Bernauer et al., 1998Go).



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FIG. 1. Biotransformation of ETBE in mammals (Bernauer et al., 1998Go).

 

    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
t-Butanol (99.5+% purity), 1,2-propane diol (99.5+% purity), 2-hydroxyisobutyrate (98+% purity), MTBE (99.8+% purity), d10-t-butanol (99+% purity), 2-hydroxy-2-methyl butyrate (98+% purity) and P2O5 (97% purity) were obtained from Aldrich Chemical Company, (Deisenhofen, Germany). ETBE (99+% purity) was obtained from Tokyo Chemical Industry Co. (Tokyo, Japan). 2-Methyl-1,2-propane diol was prepared as described (Bernauer et al., 1998Go). All other reagents and solvents were reagent grade or better and obtained from several commercial suppliers. All GC-columns were obtained from J&W scientific (Folsom, CA).

Exposure of volunteers to ETBE.
Six human volunteers (for information, see Table 1Go) were exposed to targeted concentrations of 4- and 40-ppm ETBE for 4 h in a dynamic exposure chamber (Ertle et al., 1972Go). ETBE concentrations in chamber air were determined at 15-min intervals by GC/MS. The volunteers refrained from alcoholic beverages and drugs 2 days before and throughout each experiment. Subjects did not abuse alcohol and were non-smokers or occasional smokers. Subjects were healthy as judged by medical examination and clinical blood chemistry and stated no previous occupational exposure to ETBE. Exposures started at 8 A.M. The study was carried out according to the Declaration of Helsinki, after approval by the Regional Ethical Committee of the University of Wuerzburg, Germany, and after written informed consent by the volunteers. A time interval of 4 weeks was kept between the 2 exposures. No significant differences in temperature in the chamber, number of air exchanges, or relative humidity were observed between the exposures.


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TABLE 1 Characteristics of Human Volunteers Participating in the Study
 
The design of the chamber and the generation of the chemical/air mixtures have been described previously (Ertle et al., 1972Go; Müller et al., 1972Go, 1974Go,1975Go). The chamber had a total volume of 8 m3, air-flow rate was 28 m3/h at a temperature of 22°C and a relative humidity of 50–60%. After the exposure, the urine of the volunteers was collected at given intervals for 72 h. Urine volumes were determined by the volunteers, and 2 aliquots (60 ml each) were rapidly frozen after collection and stored at –20°C until sample preparation. Metabolite concentrations in each urine sample collected were determined in duplicate.

Exposure of rats to ETBE.
Ten male (210–240 g, age 12 weeks) and 10 female (190–220 g, age 12 weeks) F344 NH rats from Harlan Winkelmann (Borchen, Germany) were exposed to targeted concentrations of 4 and 40 ppm ETBE (5 rats/sex/experiment) in the exposure chamber as described above for human volunteers. During the exposure, rats were kept separately in Macrolon® cages with free access to food and water. After the end of the exposure, the animals were transferred to metabolic cages (1 rat/cage) and urine was collected on ice for 72 h at 6-h intervals. Blood samples from the tail vein (100 µl) were taken from each rat after the end of the exposure period to quantify ETBE and t-butanol blood concentrations.

Quantification of ETBE concentration in the exposure chamber.
Samples (50 µl) of the chamber air were taken with a gas-tight syringe every 15 min. ETBE in the atmosphere of the exposure chamber was quantified by capillary gas chromatography using a Fisons 8000 gas chromatograph coupled to a Fisons MD 800 mass spectrometer. Separation was performed with a DB624 fused silica column (30 m, 0.25 mm ID; film thickness 1.4 µm) at an oven temperature of 35°C. Injector temperature was 150°C and detector temperature 200°C; split injection with a split ratio of 5:1. During the separation (run time of 5 min), the intensity of the major ion fragment in the electron impact mass spectrum of ETBE (m/z = 59) was monitored with a dwell time of 80 ms. Quantitation was based on calibration curves obtained with metered ETBE concentrations.

Quantitation of ETBE in blood.
Blood samples (10 ml) from the volunteers were taken with heparinized syringes and stored at –20°C. Volumes for blood samples from rats were 0.1 ml. Blood samples from humans (0.5 ml) and rats (0.025 ml) were transferred into GC-autosampler vials (2-ml volume for human samples and 0.2-ml volume for rat blood samples) immediately after blood sampling. The vials were capped and stored at –20°C until analysis.

For ETBE quantitation, 10 µl of an aqueous solution of MTBE (100 nmol/ml) was added through the septum, and the vials were then heated to 50°C for 1 h. ETBE concentrations were quantified by headspace GC/MS by injecting 200 µl of the headspace from the vials using split injection (split ratio of 10:1). Samples were separated using a DB-1-coated fused silica column (30 m x 0.25 mm ID, 1.0 µm film) at a temperature of 40°C using helium as carrier gas. In addition to monitoring m/z 59 (for ETBE), m/z 73 (most intensive ion fragment in the electron-impact mass spectrum of the internal standard MTBE) was monitored during the separation with dwell times of 80 ms. Quantitation was performed relative to the content of MTBE and referenced to calibration curves with fortified aliquots of blood samples from controls containing 0–20 nmol ETBE/ml blood. The method was linear in the range of concentrations used, and calibration standards were analyzed with every sample series (usually 20–30 samples). The method permitted the quantitation of 0.1 nmol ETBE/ml of blood with a signal-to-noise ratio of 5:1. When identical samples were repeatedly analyzed, deviations of the obtained quantitative results were lower than 10%. ETBE and t.-butanol concentrations reported in blood samples are the mean of a duplicate analysis of each sample.

Quantitation of t-butanol in blood.
t.-Butanol was quantified by GC/MS using d10-t-butanol as internal standard. To GC-vials (2-ml volume for human samples and 0.2-ml volume for rat blood samples) containg 0.2 ml of human blood and 0.025 ml of rat blood, 5 µl of a d10-t-butanol solution (1000 nmol/ml in water) and 160 µl of 1 N HCl for human blood and 20 µl of 1 N HCl for rat blood samples were added with a microliter syringe through the septum. The acid conditions completed cleaved t-butanol conjugates as investigated by 13C-NMR analysis of urine samples from exposures to 13C-ETBE (Bernauer et al., 1998Go). The vials were then kept at 80°C for 1 h and 200 µl of the headspace for human blood and 100 µl for rat blood samples were injected into the GC/MS using split injection (split ratio of 10 : 1). Injector and transfer line temperatures were 220°C. Samples were separated using a DB-1-coated fused silica column (30 m x 0.25 mm ID; 1.0 µm film) at a temperature of 40°C. The ions m/z 59 (t-butanol) and m/z 65 (d10-t-butanol) were monitored during the gas chromatographic separation with dwell times of 80 ms. Quantitation was performed relative to the content of d10-t-butanol and referenced to calibration curves with fortified aliquots of blood samples from controls containing 0–50 nmol/ml t-butanol. The method was linear in the range of concentrations used, and calibration standards were analyzed with every sample series (usually 20–30 samples). The method permitted the quantitation of 0.2-nmol t-butanol/ml of blood with a signal-to-noise ratio of 5:1. When identical samples were repeatedly analyzed, deviations of the obtained quantitative results were lower than 10%.

Quantitation of ETBE and ETBE-metabolites in urine.
ETBE and t-butanol in urine samples were quantified by headspace GC/MS using 0.5 ml of human urine and 0.2 ml of rat urine. ETBE and t-butanol in the urine samples were quantitated as described above for blood samples.

In addition, rat urine samples were treated with 16 µl of 32% hydrochloric acid and kept at 90°C for 15 min to cleave all t-butanol conjugates. To quantify 2-methyl 1,2-propane diol, 50 µl of a solution of the internal standard 1,2-propane diol (1000 nmol/ml in water) were added to 0.5 ml of human urine or 0.2 ml of rat urine. All urine samples were then diluted with an equivalent volume of methanol and 2-methyl 1,2-propane diol was quantified by GC/MS by injecting 1 µl of the obtained samples. Separation was achieved using a fused silica column coated with DB-FFAP (30 m x 0.32 mm; film thickness was 0.25 µm) with helium as carrier gas (2 ml/min). Samples were separated using a linear temperature program from 50°C to 230°C with a heating rate of 10°C/min. Injector and transfer line temperatures were 280°C. The concentrations of 2-methyl-1,2-propane diol were determined by monitoring m/z 59 and m/z 45 during the gas chromatographic separation with dwell times of 80 ms. Split injection (split ratio of 10:1) was used. Quantitation was performed relative to the content of 1,2-propane diol and referenced to calibration curves with fortified aliquots of urine samples from controls containing 0–200 nmol/ml 2-methyl 1,2-propane diol. The method was linear in the range of concentrations used and of calibration standards, which were analyzed with every sample series (usually 20–30 samples). The method permitted the quantitation of 1 nmol 2-methyl 1,2-propane diol/ml of urine with a signal-to-noise ratio of 5:1. When identical samples were repeatedly analyzed, deviations of the obtained quantitative results were lower than 15%.

Concentrations of 2-hydroxyisobutyrate in urine were quantified by GC/MS after transformation to the corresponding methyl ester. Urine samples (0.5 ml for humans and 0.2 ml for rats) were mixed with 2-hydroxy-2-methylbutyrate (internal standard, 100 µl of a 1000 nmol/ml solution in water). Samples were then taken to dryness using anhydrous P2O5 in an evacuated desiccator. The obtained residues were treated with 500 µl of BF3/methanol (14%) at 60°C for 30 min. Samples were then diluted with 250 µl of water and extracted with 1 ml of chloroform. The chloroform layer was dried over sodium sulfate and 2 µl of the obtained solution was analyzed by GC/MS (splitless injection). Samples were separated on a DB-WAX column (30 m x 0.25 mm; 0.25 µm film thickness) using a linear temperature program from 50°C to 230°C with a heating rate of 10°C/min. The intensities of m/z 43, 55, 49, 73, and 89 were monitored during the separation with dwell times of 80 ms. Quantitation was based on the ratio of m/z 59 to m/z 73 (internal standard). Quantitation was performed relative to the content of 2-hydroxy 2-methylbutyrate and referenced to calibration curves with fortified aliquots of urine samples from controls containg 0–1000 nmol/ml 2-hydroxyisobutyrate. The method was linear in the range of concentrations used, and a series of calibration standards (urine from controls fortified with known concentrations of 2-hydroxyisobutyrate) were analyzed with every sample series (usually 20–30 samples). The method permitted the quantitation of 3 nmol 2-hydroxyisobutyrate/ml of urine with a signal-to-noise ratio of 5:1. When identical samples were repeatedly analyzed, deviations of the obtained quantitative results were lower than 20%.

The presence of a highly variable background excretion of 2-hydroxyisobutyrate interfered with metabolite quantitation after the 4-ppm exposure. To quantitate metabolite excretions with urine over time, only samples were considered where the concentrations of 2-hydroxyisobutyrate was significantly above background. Background concentrations were determined in the urine of the volunteers and the rats before the exposure; in addition, background concentrations were quantified in urine from 4 other individuals taken in the afternoon and from 5 control rats also during the afternoon. The background was calculated as mean concentration of 2-hydroxyisobutyrate ± SD in those samples and urine samples were considered to contain increased concentrations of 2-hydroxyisobutyrate only when the determined concentrations were significantly different (p < 0.05, Student`s t-test). Values were adjusted to urine collection intervals.

GC/MS analysis.
GC/MS analyses were performed on a Fisons MD 800 mass spectrometer coupled to a Fisons 8000 GC and equipped with an AS 800 autosampler and an electron impact source (Fisons Instruments, Mainz, Germany).

Statistical analysis.
Statistical analyses of the data were performed using Student's t-test in Microsoft Excel spreadsheets. P-values less than 0.05 were considered significant. To determine possible sex-differences, all data set from the male and female animals and male and female human volunteers were compared using Student's t-test in Microsoft Excel spreadsheets.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Biotransformation of ETBE in Humans
During all experiments, the deviations between the targeted concentrations and the actual concentrations of ETBE in the chamber were less than 10% of the targeted values. Actual concentrations were 4.5 ± 0.6 ppm and 40.6 ± 3.0 ppm (mean ± SD of 16 determinations in 15-min intervals over 4 h). Experimental results on the excretion of ETBE metabolites and half-lives in humans are given in Tables 2–4GoGoGo and in Figures 2 and 3GoGo. ETBE was not detected in blood samples from the volunteers taken before exposure. The maximal concentrations of ETBE in blood were determined directly after the end of the exposure period. ETBE concentrations rapidly decreased to reach the limit of detection 24 h (40 ppm) or 4 h (4 ppm) after the end of the exposure period. The elimination process of ETBE from blood could be separated into two phases (Table 2Go). t-Butanol was detected in low concentrations (0.5 ± 0.3 nmol/ml blood) in most of the blood samples taken from the individuals before the exposure and in blood samples from control subjects. Blood samples taken from the human volunteers after exposure to 4- and 40-ppm ETBE showed statistically significant increases in t-butanol concentrations for the time period between the end of the exposure and the 12-h blood sampling after 40-ppm ETBE exposures, and between the end of the exposure and the 4-h blood sampling point after 4-ppm ETBE exposure (data not shown). The apparent half-life (h/l) of elimination of t-butanol from blood was 9.8 ± 1.4 h, and there were no statistically significant differences between the 4- and the 40-ppm exposures. The areas under the curves (AUC) for ETBE were calculated as 36.3 µmol x h/l after exposure to 40 ppm and 2.3 µmol x h/l after exposure to 4 ppm ETBE.


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TABLE 2 ETBE and t-Butanol Blood Concentrations in Humans Exposed to ETBE for 4-H
 

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TABLE 3 Cumulative Metabolite Excretion and Half-Lives of Urinary Excretion of ETBE Metabolites in Humans (n = 6) after Exposure to 40.6 ± 3.0- and 4.5 ± 0.6-ppm ETBE
 

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TABLE 4 Received Doses of ETBE in Humans and Amount of Metabolites Recovered in Urine
 


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FIG. 2. Excretion with urine of 2-hydroxyisobutyrate (filled square), 2-methyl-1,2-propane diol (filled circle) and t-butanol (filled triangle pointing up) in 6 human volunteers exposed to 40.6 ± 3.0-ppm ETBE for 4 h in a dynamic exposure chamber. Numbers (mean ± SD) given represent total amount of metabolite excreted in the urine samples collected in 6-h intervals. Each sample was analyzed in duplicate. Statistically significant differences as compared to background in controls: **, p < 0.01; *, p < 0.05.

 


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FIG. 3. Excretion with urine of 2-hydroxyisobutyrate (filled square), 2-methyl-1,2-propane diol (filled circle) and t-butanol (filled triangle pointing up) at 6 human volunteers exposed to 4.5 ± 0.6-ppm ETBE for 4 h in a dynamic exposure chamber. Numbers (mean ± SD) given represent total amount of metabolite excreted in the urine samples collected at 6-h intervals. Each sample was analyzed in duplicate. Statistically significant differences as compared to background in controls: **, p < 0.01; *, p < 0.05.

 
In urine samples of the volunteers collected before ETBE exposure and in samples collected from human controls, low concentrations of t-butanol, 2-methyl-1,2-propane diol, and 2-hydroxyisobutyrate were present. In the urine samples from exposed individuals, concentrations were significantly increased when compared to control urine samples. Statistically significant increases in the concentrations of the 3 metabolites were observed in all urine samples taken between 0 and 36 h after the end of the exposure to 40 ppm ETBE (Fig. 2Go). After 4 ppm ETBE, only few urine samples from the volunteers contained significantly increased concentrations of 2-hydroxyisobutyrate, due to the high and variable background excretion of this compound (Fig. 3Go).

Due to much lower background levels, the concentrations of 2-methyl-1,2-propane diol were significantly higher than controls in all samples collected between 0 and 36 h after exposure to 4 ppm ETBE. t-Butanol (free and conjugated) concentrations were significantly higher than controls in most samples collected between 0 and 36 h after exposure to 40 ppm ETBE and 0 and 24 h after exposure to 4 ppm ETBE (Figs. 2 and 3GoGo).

Based on the amount of 2-hydroxyisobutyrate recovered, this compound represents the major urinary metabolite of ETBE (Table 3Go), t-butanol and 2-methyl-1,2-propane diol were minor urinary metabolites of ETBE in humans.

Large variations were observed among individuals in the rates of excretion and urinary concentrations of 2-hydroxyisobutyrate. However, no statistically significant differences in the amounts of 2-hydroxyisobutyrate excreted or in the rates of excretion were seen between males and females (Table 4Go). Due to highly variable backgrounds of 2-hydroxyisobutyrate and the small increase in these concentrations due to ETBE biotransformation, the calculated metabolite excretion after 4-ppm ETBE varies widely. As with the excretion of 2-hydroxyisobutyrate, no statistically significant differences were observed between male and female volunteers in the rates of excretion or the total recovery of the other metabolites (Tables 3 and 4GoGo). Based on the sum of recovered metabolites, 2-hydroxyisobutyrate represents the major urinary ETBE-metabolite and t-butanol and 2-methyl-1,2-propane diol are minor urinary metabolites of ETBE in humans.

Biotransformation of ETBE in Rats
Rats were exposed to the same ETBE concentrations as were used in the human studies. The experimental results on metabolite concentrations and excretion are compiled in Tables 5 through 7GoGoGo and Figures 4 and 5GoGo. The concentration of ETBE in the blood of rats, determined after the end of the 4-h-exposure period, was lower than that seen in humans after identical exposure. ETBE was more rapidly cleared from rat blood than from human blood. In contrast to lower concentrations of ETBE, the determined concentrations of t-butanol were higher after both 4- and 40-ppm ETBE exposure in rats when compared to humans subjected to identical exposure concentrations.


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TABLE 5 Blood Concentrations at the End of Exposure, Background Concentrations in Non-exposed Control Rats (n = 5), and Half-Lives of Elimination from Blood after Exposure of Rats (n = 10) to 40.6 ± 3.0 and 4.5 ± 0.6 ppm ETBE for 4 H
 

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TABLE 6 Cumulative Metabolite Excretion and Half-Lives of Urinary Excretion of ETBE Metabolites in Rats (n = 10) after Exposure to 40.6 ± 3.0- and 4.5 ± 0.6-ppm ETBE
 

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TABLE 7 Received Doses of ETBE in Rats and Amount of Metabolites Recovered in Urine
 


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FIG. 4. Excretion with urine of 2-hydroxyisobutyrate (filled square), 2-methyl-1,2-propane diol (filled circle) and t-butanol (filled triangle pointing up) in 10 rats exposed to 40.6 ± 3.0-ppm ETBE for 4 h in a dynamic exposure chamber. Numbers (mean ± SD) given represent total amount of metabolite excreted in the urine samples collected at 6-h intervals. Each sample was analyzed in duplicate. Statistically significant differences as compared to background in controls: **, p < 0.01; *, p < 0.05.

 


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FIG. 5. Excretion with urine of 2-hydroxyisobutyrate (filled square) and 2-methyl-1,2-propane diol (filled circle) in 10 rats exposed to 4.5 ± 0.6-ppm ETBE for 4 h in a dynamic exposure chamber. Concentrations of t-butanol in all urine samples were not significantly increased as compared to background excretion. Numbers given represent total amount of metabolite excreted in the urine samples collected at 6-h intervals. Statistically significant differences as compared to background in controls: **, p < 0.01; *, p < 0.05.

 
t-Butanol was also detected in low concentrations in blood samples taken from control rats. Blood samples taken from rats after exposure to 4- and 40-ppm ETBE showed statistically significant increases in t-butanol concentrations after the end of the exposure (data not shown). The apparent half-life of elimination from blood of ETBE was 0.8 ± 0.2 h and the half-life of elimination of t-butanol was not determined.

In urine samples of rats collected before ETBE exposure, and in samples collected from control rats, low concentrations of t-butanol, 2-methyl-1,2-propane diol, and 2-hydroxyisobutyrate were present. In the urine samples from exposed rats, the concentrations of 2-hydroxyisobutyrate and 2-methyl-1,2-propane diol were significantly increased (as compared to controls) in urine samples collected within 24 h after the end of 40-ppm ETBE inhalation (Fig. 4Go). t-Butanol concentrations were significantly above background only between 6 and 18 h after the end of the exposure. After 4-ppm ETBE in the urine samples from exposed rats, the concentrations of 2-hydroxyisobutyrate and 1,2-methyl-1,2-propane diol were significantly increased between 6 and 18 h after exposure, but the concentrations of t-butanol were not significantly increased (Fig. 5Go).

Based on the amount of 2-hydroxyisobutyrate, this compound also represents the major urinary metabolite of ETBE (Table 6Go) in rats, t-butanol and 2-methyl-1,2-propane diol were minor urinary metabolites of ETBE.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this work, the biotransformation of ETBE was compared between rats and humans after inhalation exposure. After 4-h inhalation exposures to 4- and 40-ppm ETBE, blood levels of ETBE in humans were higher than in rats immediately after the end of the exposure period. The blood levels observed in humans in this study are similar to those seen by Nihlen et al. (1998b), who exposed humans to ETBE concentrations of 25- and 50-ppm, but only for 2 h. In addition, in contrast to our study where the volunteers were exposed at rest, the subjects in the Nihlen-study performed light work, which may result in different blood levels due to changes in lung absorption and respiratory elimination.

In general, the time course of elimination of ETBE and all metabolites quantified in this study show that rats excrete ETBE and its metabolites more rapidly than humans. In rats, ETBE from blood is rapidly cleared with a half-life of less than 1 h.

In the urine of humans and rats, the three known metabolites of ETBE excreted with urine, plus unchanged ETBE, were quantified. Due to some water solubility, a minor part of the ETBE dose is excreted unchanged with urine in humans, confirming previous information (Nihlen et al., 1998bGo). In rat urine, the ETBE concentrations in the urine samples were already below the limit of detection in the first available samples (6 h after the end of exposure).

Our study confirms the structure of ETBE metabolites formed in the rat (Bernauer et al., 1998Go) and demonstrates that identical metabolites of ETBE are formed in humans and excreted with urine. In both species, the major metabolite excreted was 2-hydroxyisobutyrate. This compound, however, was also detected in significant amounts in urine samples from all rats and all human volunteers before the ETBE exposures. 2-Hydroxyisobutyrate has been found as a urinary organic acid in humans, and it is formed endogenously (Liebich and Forst, 1984Go). The relatively high rates of excretion are unlikely to be related to exposure to chemicals (e.g., t-butanol or isoprene) metabolized to 2-hydroxyisobutyate (Bernauer et al., 1998Go; Henderson et al., 1993Go). As in rats, 2-methyl-1,2-propane diol was a minor ETBE-metabolite in humans; the presence of this compound in urine samples collected before the exposure and in control individuals is likely caused by oxidation of t-butanol. Background concentrations of t-butanol were detected in all blood samples and all urine samples from the volunteers and from other controls suggesting exposure to t-butanol from other sources besides ETBE. t-Butanol or t-butyl esters are used in food processing and flavoring (CIREP, 1989Go).

Urinary excretion of ETBE-metabolites was slower than the clearance of ETBE and t-butanol from blood; however, all ETBE-metabolites quantified in this study were eliminated in rats with apparent half-lives of elimination of less than 5 h. In humans, the elimination with urine of the metabolites formed from ETBE was considerably slower.

The extent of ETBE biotransformation in rats and in humans is similar when the amounts of metabolites and the relative concentrations recovered in urine are compared to the doses received (Tables 4 and 7GoGo). Between 41 and 53% of the calculated doses of ETBE received by inhalation were recovered as metabolites in urine, the rest of the ETBE taken up by inhalation is likely exhaled. Exhalation of unchanged ETBE was not determined in this study; however, another study using similar concentrations of ETBE has reported that exhalation is a significant pathway of elimination of ETBE. A retained percentage (net uptake rate) of 26% has been determined for exposure concentrations of up to 50 ppm (Nihlen et al., 1998aGo). In this study, based on quantitation of net uptake, clearance by inhalation, and quantitation of t-butanol and ETBE in urine, we found that about 50% of the ETBE taken up was recovered. The unaccounted 50% is likely excreted as other metabolites that were not quantified (Nihlen et al., 1998bGo).

In our study, approximately 40% of the calculated amount at a retention of 0.3 of ETBE metabolites was recovered in urine. A slightly higher recovery of the received dose (based on an identical retention in humans and rats) was observed in rats, in the form of metabolites in urine. In both species, there was an almost identical percentage of the received dose after both exposure concentrations were recovered as ETBE metabolites in urine, suggesting that a saturation of ETBE-biotransformation in the concentration range studied does not occur.

The results also confirm previous information on the intensive biotransformation of the ETBE-metabolite t-butanol (Bernauer et al., 1998Go). In previous experiments, with administration of 13C-t-butanol given orally to a human volunteer, low concentrations of t-butanol, but much higher concentrations of 2-hydroxyisobutyrate and 2-methyl-1,2-propane diol, were present in collected urine samples.

The profile of urinary metabolites was similar in rats and in humans. In both species, 2-hydroxyisobutyrate represented the major excretory product and 2-methyl-1,2-propanol and t-butanol were minor excretory products.

ETBE-biotransformation in humans is qualitatively similar to that of MTBE: identical metabolites are excreted. Due to a higher respiratory retention, higher doses of MTBE, as compared to ETBE, are received under identical exposure conditions. When comparing MTBE and ETBE biotransformation under identical exposure conditions, similar blood levels of both ethers were observed after the end of the exposure. With MTBE, the blood levels of t-butanol after exposure and the amount of metabolites recovered in urine were higher, both in rats and in humans, after both exposure concentrations, suggesting more rapid metabolism of MTBE as compared to ETBE in vivo (Amberg et al., 1999Go).

In conclusion, the biotransformation of ETBE after inhalation is qualitatively and quantitatively similar in rats and humans, and sex-differences in biotransformation were not seen in either species. Due to background exposures to t-butanol in both humans and rats, t-butanol concentrations in urine or blood may not represent the useful biomarkers of exposure to low levels of ETBE that might have been expected from environmental exposures.


    ACKNOWLEDGMENTS
 
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. Parts of this work was also supported by the Biomed Program of the European Union, Contract No. BMH4-CT96-0184.


    NOTES
 
1 To whom correspondence should be addressed. Fax: +49-0931-201-3865. E-mail: dekant{at}toxi.uni-wuerzburg.de. Back

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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Amberg, A., Rosner, E., and Dekant, W. (1999). Biotransformation and kinetics of excretion of methyl tert-butyl ether in rats and humans. Toxicol. Sci. 51, 1–8.[Abstract]

Bernauer, U., Amberg, A., and Dekant, W. (1998). Biotransformation of 12C- and 2-13C-labeled methyl tert-butyl ether, ethyl tert-butyl ether, and tert-butanol in rats: Identification of metabolites in urine by 13C nuclear magnetic resonance and gas chromatographymass spectroscopy. Chem. Res. Toxicol. 11, 651–658.

Bird, M. G., Burleigh-Flayer, H. D., Chun, J. S., Douglas, J. F., Kneiss, J. J., and Andrews, L. S. (1997). Oncogenicity studies of inhaled methyl tertiary-butyl ether (MTBE) in CD-1 mice and F344 rats. J. Appl. Toxicol. 17, S45–S55.[ISI][Medline]

Borghoff, S. J., Prescott-Mathews, J. S., and Poet, T. S. (1996). The mechanism of male rat kidney tumors induced by methyl tert-butyl ether and its relevance in assessing human risk. CIIT Activities 16, 1–8.

Burleigh-Flayer, H. D., Chun, J. S., and Kintigh, W. J. (1992). Methyl tertiary butyl ether: Vapor inhalation oncogenicity study in CD-1 mice. BRRC Project Number 91NO013A, Bushy Bun Research Center, Export, PA.

CIREP (Cosmetic Ingredient Review Expert Panel) (1989). Final report on the safety assessment of t-butyl alcohol. J. Am. Coll. Toxicol 8, 627–641.

Costantini, M. G. (1993). Health effects of oxygenated fuels. Environ. Health Perspect. 6, 151–160.

Donoghue, W. C. (1985). How to measure your % body-fat. Creative Health Products, Plymouth, MI.

Duffy, J. S., Del Pup, J. A., and Kneiss, J. J. (1992). Toxicological evaluation of methyl tertiary butyl ether (MTBE): Testing performed under TSCA consent agreement. J. Soil Contam. 1, 29–37.

ECETOC (1997). Methyl tert-butyl ether (MTBE) health risk characterization. ECETOC Technical Report No. 72 (CAS No. 1634-04-4) 1–67.

Ertle, T., Henschier, D., Müller, G., and Spassowski, M. (1972). Metabolism of trichloroethylene in man: 1. The significance of trichloroethanol in long-term exposure conditions. Arch. Toxikol. 29, 171–188.[ISI][Medline]

Henderson, R. F., Sabourin, P. J., Bechtold, W. E., Steinberg, B., and Chang, Y. 1993). Disposition of inhaled isobutene in F344/N rats. Toxicol. Appl. Pharmacol. 123, 50–61.

Liebich, H. M., and Forst, C. (1984). Hydroxycarboxylic and oxocarboxylic acids in urine: Products from branched-chain amino acid degradation and from ketogenesis. J. Chromatogr. 309, 225–242.[Medline]

Müller, G., Spassowski, M., and Henschler, D. (1972). Trichloroethylene exposure and trichloroethylene metabolites in urine and blood. Arch. Toxikol. 29, 335–340.[ISI][Medline]

Müller, G., Spassowski, M., and Henschier, D. (1974). Metabolism of trichloroethylene in man: II. Pharmacokinetics of metabolites. Arch. Toxikol. 32, 283–295.

Müller, G., Spassowski, M., and Henschier, D. (1975). Metabolism of trichloroethylene in man: III. Interaction of trichloroethylene and ethanol. Arch. Toxikol. 33, 173–189.

Nihlen, A., Löf, A., and Johanson, G. (1998a). Controlled ethyl tert-butyl ether (ETBE) exposure of male volunteers: 1. Toxicokinetics. Toxicol. Sci. 46, 1–10.[Abstract]

Nihlen, A., Löf, A., and Johanson, G. (1998b). Experimental exposure to methyl tert-butyl ether: I. Toxicokinetics in humans. Toxicol. Appl. Pharmacol. 148, 274–280.[ISI][Medline]

Rudo, K. M. (1995). Methyl tertiary butyl ether (MTBE)—evaluation of MTBE carcinogenicity studies. Toxicol. Ind. Health 11, 167–173.[ISI][Medline]

Scientific Advisory Board on Toxic Air Pollutants, S. (1995). Summary of the carcinogenicity assessment of MTBE. Environ. Health Perspect. 103, 420–422.

Swenberg, J. A., Short, B., Borghoff, S., Strasser, J., and Charbonneau, M. (1989). The comparative pathobiology of {alpha}-globulin nephropathy. Toxicol. Appl. Pharmacol. 97, 35–46.[ISI][Medline]

Takahashi, K., Lindamood, C., and Maronpot, R. R. (1993). Retrospective study of possible {alpha}-globulin nephropathy and associated cell proliferation in male Fischer 344 rats dosed with t-butyl alcohol. Environ. Health Perspect. 101, 281–285.[ISI][Medline]

White, R. D., Daughtry, W. C., and Wells, M. S. (1995). Health effects of inhaled tertiary amyl ether and ethyl tertiary butyl ether. Toxicol. Lett. 82/83, 719–724.