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

Transcription

1 TOXICOLOGICAL SCIENCES 51, 1 8 (1999) Copyright 1999 by the Society of Toxicology Biotransformation and Kinetics of Excretion of Methyl-tert-Butyl Ether in Rats and Humans Alexander Amberg, Elisabeth Rosner, and Wolfgang Dekant 1 Institut für Toxikologie, Universität Würzburg, Versbacher Str. 9, Würzburg, Germany Received January 12, 1999; accepted March 31, 1999 Methyl-tert-butyl ether (MTBE) is widely used as an additive to gasoline to increase oxygen content and reduce tail pipe emission of pollutants. Therefore, widespread human exposure may occur. To contribute to the characterization of potential adverse effects of MTBE, its biotransformation was compared in humans and rats after inhalation exposure. Human volunteers (3 males and 3 females) and rats (5 each, males and females) were exposed to 4 ( ) and 40 ( ) ppm MTBE for 4hinadynamic exposure system. Urine samples from rats and humans were collected for 72 h in 6-h intervals, and blood samples were taken in regular intervals for 48 h. In urine, MTBE and the MTBE metabolites tertiary-butanol (t-butanol), 2-methyl-1,2-propane diol, and 2-hydroxyisobutyrate were quantified; MTBE and t-butanol were determined in blood samples. After the end of the exposure period, inhalation of 40 ppm MTBE resulted in blood concentrations of MTBE M in rats and M in humans. The MTBE blood concentrations after inhalation of 4 ppm MTBE were in rats and M in humans. MTBE was rapidly cleared from blood with a half-life of h in humans and h in rats. The blood concentrations of t-butanol were M in humans and M in rats after 40 ppm MTBE, and in humans and in rats after 4 ppm MTBE. In humans, t-butanol was cleared from blood with a half-life of h. 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 urine of both humans and rats exposed to MTBE, the concentrations of these compounds were significantly increased. 2-Hydroxyisobutyrate was recovered as a major excretory product in urine; t-butanol and 2-methyl-1,2- propane diol were minor metabolites. All metabolites of MTBE excreted with urine were rapidly eliminated in both species after the end of the MTBE exposure. Elimination half-lives for the different urinary metabolites of MTBE were between 7.8 and 17.0 h in humans and 2.9 to 5.0 h in rats. The obtained data indicate that MTBE biotransformation and excretion are similar in rats and humans, and MTBE and its metabolites are rapidly excreted in both species. Between 35 and 69% of the MTBE retained after the end of the exposure was recovered as metabolites in urine of both humans and rats. 1 To whom all correspondence should be addressed. Fax: dekant@toxi.uni-wuerzburg.de. Key Words: methyl-tert-butyl ether (MTBE); tert-butanol (tbutanol); inhalation exposure; excretion; rat; human. The presence of oxygen-containing compounds to reduce harmful engine emissions from cars 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, 1993). Methyl tertiary (tert)- butyl ether (MTBE) and ethanol are the major oxygenates presently in use. Due to possible widespread exposure of humans to these ethers when refueling cars or while commuting in heavy traffic, several programs are underway to study the toxicology of these compounds (White et al., 1995). The acute toxicity of MTBE is low. Both MTBE and its metabolite tert-butanol (t-butanol) have been studied in longterm bioassays for tumorigenicity (for review, see Bird et al., 1997; ECETOC, 1997; Rudo, 1995). MTBE and t-butanol induce renal tumors in male rats (Burleigh-Flayer et al., 1992; Takahashi et al., 1993). Renal tumor induction by these compounds may be mediated by the accumulation of 2u -globulin (Borghoff et al., 1996; Scientific Advisory Board on Toxic Air Pollutants, 1995). An impaired degradation of this protein induced by bound metabolites of t-butanol and/or MTBE may cause renal toxicity, cell proliferation, and finally renal tumors (Swenberg et al., 1989). MTBE exposure also increased the incidence of liver tumors in female mice and testicular tumors in male rats (Belpoggi et al., 1995). Testicular tumors in male rats were also observed following oral administration of MTBE (Burleigh-Flayer et al., 1992). MTBE and its metabolite t- butanol are negative in standard genotoxicity studies (Duffy et al., 1992). As a part of the effort to characterize the toxic effects of MTBE, we investigated the biotransformation and toxicokinetics of MTBE in rats and in human volunteers. Intensive metabolism of MTBE 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 MTBE and a high percentage of retention of inhaled MTBE (Johanson et al., 1995). In rats exposed to MTBE by inhalation, t-butanol, 2-methyl 1,2-pro- 1

2 2 AMBERG, ROSNER, AND DEKANT FIG. 1. Biotransformation of MTBE in mammals. pane diol and 2-hydroxyisobutyrate were excreted as metabolites in urine (Fig. 1). 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., 1998). MATERIALS AND METHODS Chemicals. MTBE (99.8 % purity), t-butanol (99.5 % purity), 2-hydroxyisobutyrate (98 % purity), ethyl tert-butyl ether (99 % purity), d 10 -tbutanol, 2-hydroxy-2-methyl butyrate (98 % purity), and P 2 O 5 (97% purity) were obtained from Aldrich Chemical Company (Deisenhofen, Germany). 2-Methyl-1,2-propane diol was prepared as described (Bernauer et al., 1998). 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 MTBE. Three healthy females (ages years, body weights between 50 and 58 kg) and 3 healthy male volunteers (ages years, body weights between 75 and 84 kg) were exposed to targeted concentrations of 4 and 40 ppm MTBE for 4hinadynamic exposure chamber (Ertle et al., 1972). MTBE concentrations in chamber air were determined at 15-min intervals by GC/MS. The volunteers had to refrain 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, stated no previous occupational exposure to MTBE, and did not refuel their cars during the 2 days prior to exposure and during sample collection period. Exposures started at 8 A.M. The study was carried out according to the Declaration of Helsinki, after approval by the Regional Ethical Commitee 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. The design of the chamber and the generation of the chemical/air mixtures has been described previously (Ertle et al., 1972; Müller, G. et al., 1972, 1974, 1975). The chamber had a total volume of 8 m 3, air flow rate was 28 m 3 /h at a temperature of 22 C, and a relative humidity was maintained 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 two 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 MTBE. Five male ( g, aged 12 weeks) and five female ( g, aged 12 weeks) F344 NH rats from Harlan Winkelmann (Borchen, Germany) were exposed to targeted concentrations of 4 and 40 ppm MTBE 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 and urine was collected on ice for 72 h in 6-h intervals. Blood samples from the tail vein (100 l) were taken from each rat after the end of the exposure period to quantify MTBE and t-butanol blood concentrations. Quantification of MTBE concentration in the exposure chamber. Samples (50 l) of the chamber air were taken every 15 min with a gas-tight syringe. MTBE 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 I.D., film thickness 1.4 m) 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 fragment ion in the electron impact mass spectrum of MTBE (m/z 73) was monitored with a dwell time of 80 ms. Quantitation was based on calibration curves obtained with metered MTBE-concentrations. Quantitation of MTBE 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 100 l. Blood samples from humans (0.5 ml) and rats (0.025 ml) were transferred into GC-autosampler vials (2-ml volume for both human and rat blood samples) immediately after blood sampling. The vials were capped and stored at 20 C until analysis. For MTBE-quantitation, 10 l of an aqueous solution of ethyl tert-butyl ether (100 nmol/ml) was added through the septum, and the vials were then heated to 50 C for 1 h. MTBE concentrations were quantified by headspace GC/MS by injecting 200 l 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 0.25 mm ID, 1.0 m film) at a temperature of 40 C. In addition to monitoring m/z 73 (for MTBE), m/z 59 (most intensive fragment ion in the electron impact mass spectrum of the internal standard ETBE) was monitored during the separation with dwell times of 80 ms. Quantitation was performed relative to the content of ETBE and referenced to calibration curves with fortified aliquots of blood samples from controls containing 0 10 nmol MTBE/ml blood. The method was linear in the range of concentrations used, and calibration standards were analyzed with every sample series (usually samples). The method permitted the quantitation of 0.1 nmol MTBE/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%. MTBE and t-butanol concentrations reported in blood samples are based on duplicate analysis. Quantitation of t-butanol in blood. t-butanol was quantified by GC/MS using d 10 -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 ml of rat blood, 5 lofad 10 -t-butanol solution (1000 nmol/ml in water), and 160 l of 1 N HCl for human blood and 20 l of 1 N HCl for rat blood samples were added with a microliter syringe through the septum. The vials were then kept at 80 C for 1 h, and 200 l of the headspace for human blood and 100 l 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 0.25 mm ID, 1.0 m film) at a temperature of 40 C. The ions m/z 59 (t-butanol) and m/z 65 (d 10 -t-butanol) were monitored during the gas chromatographic separation with dwell times of 80 msec. Quantitation was performed relative to the content of d 10 -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 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

3 MTBE BIOTRANSFORMATION 3 repeatedly analyzed, deviations of the obtained quantitative results were lower than 10%. Quantitation of MTBE and MTBE metabolites in urine. MTBE 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. MTBE and t-butanol in the urine samples was quantitated as described above for blood samples. t-butanol conjugates in urine were cleaved by the acid treatment to t-butanol. To quantify 2-methyl 1,2-propane diol, 50 l 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. In addition, rat urine samples were treated with 16 l of 32% hydrochloric acid and kept at 90 C for 15 min. 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 l ofthe obtained samples. Separation was achieved using a fused silica column coated with DB-FFAP (30 m 0.32 mm, film thickness 0.25 m) 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. 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 nmol/ml 2-methyl 1,2-propane diol. The method was linear in the range of concentrations used and calibration standards were analyzed with every sample series (usually 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 l of a 1000-nmol/ml solution in water). Samples were then taken to dryness using anhydrous P 2 O 5 in an evacuated desiccator. The obtained residues were treated with 500 l ofbf 3 /methanol (14%) at 60 C for 30 min. Samples were then diluted with 250 l of water and extracted with 1 ml of chloroform. The chloroform layer was dried over sodium sulfate and 2 l of the obtained solution was analyzed by GC/MS (splitless injection). Samples were separated on a DB-WAX column (30 m 0.25 mm, 0.25 m film thickness) using a linear temperature program from 50 C to 230 C with a heating rate of 10 C/min. The intensities of mz 43, 55, 49, 73, and 89 were monitored during the separation with dwell times of 80 msec. Quantitation was TABLE 1 Blood Concentrations at the End of Exposure, Background Concentrations in Non-exposed Controls, and Half-lives of Elimination from Blood after Exposure of Humans and Rats to ppm MTBE for 4 H Concentration ( M) Background ( M) Half-life (h) Human MTBE nd t-butanol ** Rat MTBE nd t-butanol ** Not determined Note. Numbers represent mean SD; nd, not detected. Non-exposed controls, n 6 for humans and n 5 for rats. Exposed humans, n 6; exposed rats, n 10. *Statistically significant difference from background levels (p 0.05). **Statistically significant difference from background levels (p 0.01). TABLE 2 Blood Concentrations at the End of Exposure, Background Concentrations in Non-exposed Controls and Half-lives of Elimination from Blood after Exposure of Humans and Rats to ppm MTBE for 4 H Concentration ( M) Background ( M) Half-life (h) Human MTBE nd t-butanol ** Rat MTBE nd Not determined t-butanol * Not determined Note. Numbers represent mean SD; nd, not detected. Non-exposed controls, n 6 for humans; n 5 for rats. Exposed humans, n 6, exposed rats, n 10. *Statistically significant difference from background levels (p 0.05). **Statistically significant difference from background levels (p 0.01). 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 nmol/ml 2-hydroxyisobutyrate. The method was linear in the range of concentrations used and calibration standards were analyzed with every sample series (usually 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 18%. 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 Biotransformation of MTBE in Humans During all experiments, the deviations between the targeted concentrations and the actual concentrations of MTBE in the chamber were less than 10% of the targeted values. Actual concentrations were ppm and ppm (mean SD of 16 determinations in 15-min intervals over 4 h). Experimental results on the excretion of MTBE metabolites and half-lives in humans are given in Tables 1, 2, 3, and 4 and in Figures 2 and 3. MTBE was not detected in blood samples from the volunteers taken before exposure. The maximal concentrations of MTBE in blood were determined directly after the end of the exposure period; MTBE concentrations rapidly decreased to reach the limit of detection 24 h (40 ppm) or 8h(4ppm) after the end of the exposure period. t-butanol was detected in low concentrations ( nmol/ml blood) in most of the blood samples taken from the

4 4 AMBERG, ROSNER, AND DEKANT TABLE 3 Cumulative Metabolite Excretion and Half-lives of Urinary Excretion of MTBE Metabolites in Humans and rats after Exposure to ppm MTBE Human Rat Total excretion Background Half-life (h) Total excretion Background Half-life (h) MTBE nd nd nd t-butanol * * Methyl-1,2-propane diol * * Hydroxyisobutyrate * * Note. Metabolite amounts recovered are calculated by using only urine samples with metabolite concentrations significantly above background excretion (Figs. 2 and 4). Background levels given were adjusted for sample collection periods. Background levels were determined in all volunteers and rats before the exposure and in control subjects (n, 6) and rats (n, 10). In rats, MTBE excretion with urine was observed, the concentrations were, however, close to the limit of detection and therefore not quantified. Numbers represent mean SD; nd, not detected. Humans, n 6; rats, n 10. *Statistically significant difference from background levels (p 0.01). individuals before exposure and in blood samples from control subjects. Blood samples taken from the volunteers after exposure to 4 or 40 ppm MTBE showed statistically significant increases in t-butanol concentrations. These increases were observed for the time period between the end of the exposure and the 12-h blood sampling after 40 ppm MTBE and between the end of the exposure and the 6-h blood sampling point after 4 ppm MTBE (data not shown). In urine samples of the volunteers, collected before MTBE exposure, and in samples collected from controls, low concentrations of t-butanol, 2-methyl-1,2-propane diol and 2-hydroxyisobutyrate were present. In the urine samples from exposed individuals, these concentrations were significantly increased as 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 48 h after the end of the exposure to 40 ppm MTBE (Fig. 2). After 4 ppm MTBE, only a few urine samples from the volunteers contained significantly increased concentrations of 2-hydroxyisobutyrate due to the high background of this compound (Fig. 3). 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 42 h after exposure to 4 ppm MTBE. t-butanol (free and conjugated) concentrations were significantly higher than controls in most samples collected between 0 and 48 h after exposure to 40 ppm MTBE and 0 and 30 h after exposure to 4 ppm MTBE (Fig. 2 and 3). Based on the amount of 2-hydroxyisobutyrate recovered, this compound represents the major urinary metabolite of MTBE (Tables 3 and 4), t-butanol and 2-methyl-1,2-propane diol were minor urinary metabolites of MTBE in humans. TABLE 4 Cumulative Metabolite Excretion and Half-lives of Urinary Excretion of MTBE Metabolites in Humans and Rats after Exposure to ppm MTBE Human Rat Total excretion Background Half-life (h) Total excretion Background Half-life (h) MTBE nd nd nd t-butanol ** * Methyl-1,2-propane diol ** ** Hydroxyisobutyrate ** Note. Metabolite amounts recovered are calculated by using only urine samples with metabolite concentrations significantly above background excretion (Figs. 3 and 5). Background levels given were adjusted for sample collection periods. Background levels were determined in all volunteers and rats before the exposure and in control subjects (n, 6) and rats (n, 10). In rats, MTBE excretion in urine was observed; the concentrations were, however, close to the limit of detection and therefore not quantified. Numbers represent mean SD; nd, not detected. Humans, n 6 in control and study groups; rats, n 10 in control and study groups. *Statistically significant difference from background levels (p 0.05). **Statistically significant difference to background levels (p 0.01).

5 MTBE BIOTRANSFORMATION 5 FIG. 2. Excretion in urine of 2-hydroxyisobutyrate ( ), 2-methyl-1,2- propane diol (F), and t-butanol (Œ) in 6 human volunteers exposed to ppm MTBE for 4hinadynamic exposure chamber. Numbers (mean SD) given represent total amounts 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 FIG. 4. Excretion in urine of 2-hydroxyisobutyrate ( ), 2-methyl-1,2- propane diol (F), and t-butanol (Œ) in 10 rats exposed to ppm MTBE for 4hinadynamic exposure chamber. Numbers (mean SD) given represent total amounts 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 FIG. 3. Excretion in urine of 2-hydroxyisobutyrate ( ), 2-methyl-1,2- propane diol (F), and t-butanol (Œ) in 6 human volunteers exposed to ppm MTBE for 4hinadynamic exposure chamber. Numbers (mean SD) given represent total amounts 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). Large variations between individuals in the rates of excretion and the urinary concentrations of 2-hydroxyisobutyrate were observed; however, no statistically significant differences in the amounts of 2-hydroxyisobutyrate excreted or in the rates of excretion were seen between males and females. As with the excretion of 2-hydroxyisobutyrate, statistically significant differences in the rates of excretion or the total recovery of the other metabolites between male and female volunteers were not observed. The determined half-lives of elimination in urine also were not significantly different between the 4- and 40- ppm-mtbe exposures (Tables 3 and 4). Based on the sum of recovered metabolites, 2-hydroxyisobutyrate represent the major urinary MTBE metabolite, and t-butanol and 2-methyl-1,2- propane diol are minor urinary metabolites of MTBE in humans. Biotransformation of MTBE in Rats Rats were exposed to the same MTBE-concentrations as used in the human studies. The experimental results on metabolite concentrations and excretion are compiled in Tables 3 and 4. The concentrations of MTBE in blood of rats determined after the end of the 4-h-exposure period were similar to those seen in humans after identical exposure concentrations. MTBE was more rapidly cleared from rat blood when compared to human blood. The determined concentrations of t-butanol were also in the same order of magnitude, both after 4- and 40-ppm MTBE exposures in rats, compared to humans subjected to identical exposure concentrations. t-butanol was also detected in low concentrations in blood samples taken from control rats. Blood samples taken from rats after exposure to 4 or 40 ppm MTBE showed statistically significant increases in t-butanol concentrations for the time after the end of the exposure (data not shown). In urine samples of rats collected before MTBE 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 (compared to controls) in urine samples collected within 24 h after the end of 40-ppm- MTBE inhalation (Fig. 4). t-butanol concentrations were significantly above background only 6 and 12 h after the end of the exposure. After 4 ppm MTBE only a few urine samples contained significantly increased concentrations of 2-hydroxyisobutyrate and t-butanol (Fig. 5). Due to much lower back-

6 6 AMBERG, ROSNER, AND DEKANT FIG. 5. Excretion in urine of 2-hydroxyisobutyrate ( ), 2-methyl-1,2- propane diol (F), and t-butanol (Œ) in 10 rats exposed to ppm MTBE for4hinadynamic exposure chamber. 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). ground levels, the concentrations of 2-methyl-1,2-propane diol were significantly higher than controls in all samples collected between 0 and 24 h after exposure to 4 ppm MTBE. Based on the amount of 2-hydroxyisobutyrate, this compound also represents the major urinary metabolite of MTBE (Tables 3 and 4) in rats; t-butanol and 2-methyl-1,2-propane diol were minor urinary metabolites of MTBE. DISCUSSION In this work, the biotransformation of MTBE in rats and in humans was compared after inhalation exposure. After 4-h inhalation exposures to 4 and 40 ppm MTBE, similar blood levels of MTBE were found in both humans and rats immediately after the end of exposure period. The blood levels observed in humans in this study are similar to those seen by Nihlen et al. (1998), who exposed humans to lower MTBE concentrations. However, in contrast to our study where the volunteers were exposed at rest, the subjects in the Nihlen study performed light work, which likely resulted in higher blood levels due to increased rates of respiration and increased blood flow through the lung. Similar blood levels of MTBE were also seen in other studies exposing humans by inhalation, after correction for exposure levels and exposure times (Prah et al., 1994; Cain et al., 1996; Pekari et al., 1996). In general, the time course of elimination of MTBE and all metabolites quantified in this study shows that rats excrete MTBE and its metabolites more rapidly than humans. In rats, MTBE from blood is rapidly cleared with a half-life of 30 min in agreement with other studies. Half-lives of elimination of MTBE from blood determined in other studies were also 0.5 h in rats (Miller et al., 1997) and between 2 and 4hinhumans (Prah et al., 1994; Nihlen et al., 1998). t-butanol is also rapidly cleared from human blood after inhalation exposure. As in other studies, no sex-differences in the apparent half-lives of elimination of these compounds were seen. In urine of humans and rats, the three known metabolites of MTBE excreted with urine and unchanged MTBE were quantified. Due to some water solubility, a minor part of the MTBE dose is excreted with urine in humans, confirming previous information (Nihlen et al., 1998). In rat urine, the MTBE concentrations in the urine samples were already (6 h after the end of exposure) below the limit of detection in the first available samples. The study confirms the structure of MTBE metabolites formed in the rat (Bernauer et al., 1998) and demonstrates that identical metabolites of MTBE are formed in humans and excreted with urine. In both species, the major metabolite of MTBE excreted with urine is 2-hydroxyisobutyrate. This compound, however, was also detected in significant amounts in urine samples from all rats and all human volunteers before the MTBE exposures. 2-Hydroxyisobutyrate has been found as a urinary organic acid in humans, and it is formed endogenously (Liebich and Forst, 1984). The relatively high rates of excretion are unlikely to be related to exposure to chemicals (tbutanol, isoprene) metabolized to 2-hydroxyisobutyate (Bernauer et al., 1998; Henderson et al., 1993). As in rats, 2-methyl-1,2-propane diol was a minor MTBE-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 than MTBE. t-butanol or t-butyl esters are used in food processing and flavoring (CIREP, 1989). Urinary excretion of MTBE-metabolites was slow compared to that via exhalation. However, all MTBE-metabolites quantified in this study were eliminated in rats with apparent halflives of elimination of less than 5 h; in humans, the elimination with urine of the metabolites formed from MTBE was considerably slower. The extent of MTBE-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 (Table 5). Between 35 and 69% of the calculated doses of MTBE received by inhalation was recovered as metabolites in urine; the rest of the MTBE taken up by inhalation is likely exhaled. Exhalation of unchanged MTBE was not determined in this study; however, several other studies using similar concentrations of MTBE have reported that exhalation is a significant pathway of elimination of MTBE. A retained percentage (net uptake rate) of 40% has been determined for exposure concentrations of up to 74 ppm (Riihimaki et al., 1996). This observation is consistent with the metabolic clearance reported for MTBE (Nihlen et al., 1998). In this study, based on quantitation of net uptake, clearance by inha-

7 MTBE BIOTRANSFORMATION 7 TABLE 5 Received Doses of MTBE in Humans and Rats and Amount of Metabolites Recovered in Urine MTBE concentration in chamber Dose received (mmol) of excreted metabolites a (mmol) Metabolite excretion a (% of received dose) Human Rat Human Rat Human Rat Note. Received doses were calculated based on a human alveolar ventilation rate of 9 L/min and a retention of 0.4; a rat alveolar ventilation rate of L/min and a retention of 0.5 (ECETOC, 1997). a Background corrected. lation, and quantitation of t-butanol and MTBE in urine, 33 48% of the MTBE taken up was recovered. The unaccounted 67 52% is likely excreted as metabolites which were not quantified (Nihlen et al., 1998). In rats exposed to 400 ppm MTBE for 6 h, more than 60% of the received dose of MTBE was recovered (Miller et al., 1997). The results also confirm previous information on the intensive biotransformation of the MTBE-metabolite t-butanol (Bernauer et al., 1998). In previous experiments with administration of 13 C-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. In conclusion, the biotransformation of MTBE after inhalation is qualitatively and quantitatively similar in rats and humans, 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 useful biomarkers of exposure to low levels of MTBE, as expected from environmental exposure. ACKNOWLEDGMENTS Research described 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 ) and certain motor vehicle and engine manufacturers. 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