Institut für Toxikologie, Universität Würzburg, Versbacher Str. 9, 97078 Würzburg, Germany
Received November 16, 1999; accepted February 4, 2000
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ABSTRACT |
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Key Words: biotransformation; F344 NH rats; humans; inhalation exposure; tert-Amyl methyl ether (TAME).
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INTRODUCTION |
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Due to the potential widespread exposure of humans to oxygenates in fuel (HEI, 1996; Vainiotalo et al., 1999), studies on the toxicity of oxygenates including TAME are underway. The major toxic effect seen in rodents after inhalation of high concentrations of TAME is central nervous system depression. Subchronic inhalation studies with TAME for 4 weeks (6 h per day, 5 days per week) showed significantly reduced body weights and relative increases in adrenal, kidney, testes, brain, and lung weights in male rats exposed to 4000 ppm TAME, but no treatment-related histopathologic findings (White et al., 1995
). The toxicity of the initial metabolite formed from TAME, tert-amyl alcohol (t-amyl alcohol), is also low. In rats exposed to concentrations of up to 1000 ppm TAME for 90 days by inhalation, increased liver weights in male rats were the only effect observed (Nolan et al., 1976
, 1981
).
The biotransformation of TAME has been studied in rats and humans, and metabolites have been identified (Fig. 1). Free and glucuronidated 2-methyl-2,3-butane diol and a glucuronide of t-amyl alcohol were major urinary metabolites excreted in rats; 2-hydroxy-2-methyl-butyric acid and 3-hydroxy-3-methyl-butyric acid were minor excretory products formed from TAME. In humans, 2-hydroxy-2-methylbutyric acid and 3-hydroxy-3-methylbutyric acid were major excretory products in addition to 2-methyl-2,3-butane diol (Amberg et al., 1999a
). These results suggest further intensive biotransformation of t-amyl alcohol by metabolic oxidation reactions.
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MATERIALS AND METHODS |
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Exposure of volunteers to TAME.
Three healthy female and three healthy male volunteers (Table 1) were exposed to targeted concentrations of 4 and 40 ppm TAME for 4 h in a dynamic exposure chamber (Ertle et al., 1972
). TAME concentrations in chamber air were determined at 15-min intervals by GC/MS. Actual TAME concentrations were 3.8 ± 0.2 and 38.4 ± 1.7 ppm. The volunteers had to refrain from alcoholic beverages and drugs 2 days before and throughout each experiment. Subjects did not abuse alcohol and were nonsmokers or occasional smokers. Subjects were healthy as judged by medical examination and clinical blood chemistry, stated no previous occupational exposure to TAME, and did not refuel their cars during the 2 days prior to exposure and during sample collection period. Exposures started at 8 am. 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 two exposures. No significant differences in temperature in the chamber, number of air exchanges, and relative humidity were observed between the exposures.
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Exposure of rats to TAME.
Five male (210230 g, 12 weeks of age) and five female (190210 g, 12 weeks of age) F344 NH rats from Harlan-Winkelmann (Borchen, Germany) were exposed to targeted concentrations of 4 and 40 ppm TAME 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 cages were checked for urine and feces, 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 µl) were taken from each rat after the end of the exposure period to quantify TAME and t-amyl alcohol blood concentrations.
Quantification of TAME concentration in the exposure chamber.
Samples (50 µl) of the chamber air were taken every 15 min with a gas-tight syringe. TAME 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 DB-1 fused silica column (30 m, 0.25 mm I.D., film thickness 1 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 fragment ion in the electron impact mass spectrum of TAME (m/z = 73) was monitored, with a dwell time of 80 msec. Quantitation was based on calibration curves obtained with metered TAME concentrations.
Quantitation of TAME and t-amyl alcohol in blood.
Blood samples (10 ml) from the volunteers were taken with heparinized syringes. Volumes for blood samples from rats were 100 µl. 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 to avoid loss of the volatile analytes during manipulations. The vials were capped and stored at 20°C for a maximum of 4 weeks.
For TAME and t-amyl alcohol quantitation, 5 µl of an aqueous solution of the internal standard tert-butanol (1000 nmol/ml) was added through the septum with a microliter syringe, and the vials were then heated to 70°C for 1 h. TAME and t-amyl alcohol-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. In addition to monitoring m/z 73 (for TAME), m/z 59 (most intensive fragment ion in the electron impact mass spectrum of t-amyl alcohol and the internal standard t-butanol) were monitored during the separation, with dwell times of 80 msec. Quantitation was performed relative to the content of t-butanol and referenced to calibration curves with fortified aliquots of blood samples from controls containing 020 nmol TAME and 020 nmol t. amyl alcohol/ml blood. The method was linear in the range of concentrations used and calibration standards were analyzed with every sample series (usually 1020 samples) The method permitted the quantitation of 0.1 nmol TAME and 0.2 nmol t-amyl alcohol/ml of blood with a signal-to-noise ratio of 5:1. When identical samples were repeatedly analyzed, the coefficients of variation were lower than 10% (n = 8). TAME and t-amyl alcohol concentrations reported in blood samples are based on duplicate analysis of samples from every individual.
Quantitation of TAME and TAME metabolites in urine.
TAME and free t-amyl alcohol in urine samples were quantified by headspace GC/MS using 0.5 ml of human urine and 0.2 ml of rat urine. TAME and t-amyl alcohol in the urine samples was quantified as described above for blood samples. Two different methods were used to quantify t-amyl alcohol and 2-methyl-2,3-butane diol glucuronides. The first method involves direct analysis of the glucuronides by GC/MS-determination of trimethylsilyl derivatives. To quantify glucuronide excretion, 50 µl of a 1000-nmol/ml solution of the internal standard glucuronic acid were added to 100 µl of human or rat urine, and the mixtures were lyophilized. The obtained residues were treated for 30 min with 1 ml of a mixture of hexamethyldisilazane, trimethylchlorosilane solution, and pyridine (2:1:9, v:v:v) at 80°C in a closed reaction vial. From the obtained solution, 2 µl were injected into the GC/MS. Separation was performed using a DB-1 coated fused silica column (30 m x 0.25 mm ID, 1-µm film). Injector temperature was 310°C, and transfer line temperature was 310°C. Samples were injected using split injection (split ratio of 10:1); oven temperature was 100°C and increased to 310°C with a rate of 10°C/min. Samples were monitored using m/z 204 and 217. Quantitation was performed relative to the content of glucuronic acid and referenced to calibration curves with fortified aliquots of urine samples from controls containing 01000 nmol/ml of the glucuronides isolated from urine of TAME-treated rats by prep HPLC (Amberg et al., 2000). This method was not very sensitive and could be used only with samples containing high concentrations of the glucuronides. In addition, the injected mixture resulted in a rapid deterioration of the performance of the mass spectrometer. Therefore, all samples were analyzed by a simpler and more sensitive method using acid hydrolysis of the glucuronides. Enzymatic hydrolysis was not very effective with reference compounds. Glucuronidase did not completely cleave the glucuronides within 24 h. Under the conditions of the acid hydrolysis, the alcohols formed by the acid hydrolysis were further converted by an acid-catalyzed dehydration of t-amyl alcohol to give 2-methyl-2-butene and of 2-methyl-2,3-butane diol to form 3-methyl-2-butanone. The efficiency of the acid hydrolysis and the dehydration was checked by NMR using urine samples from rats treated with 13C-TAME (Amberg et al., 1999a
) monitoring the disappearance of the glucuronide signals. To quantify content of free alcohols and glucuronides, t-butanol (25 µl from a 1000-nmol/ml solution in water; t-butanol is cleaved to 2-methylpropene under acidic conditions) as internal standard and 60 µl of 10 M sulfuric acid were added to 200 µl of urine in a closed vial. After 1 h at 90°C, 500 µl of the gas phase from the vial was analyzed by GC/MS. Separation was performed using a DB-1 coated fused silica column (30 m x 0.25 mm ID, 1-µm film). Samples were injected using splitless injection; oven temperature was 40° C. Samples were monitored using m/z 56, 70, and 86 by selected ion monitoring. Quantitation was performed relative to the formed 2-methylpropene and referenced to calibration curves with fortified aliquots of urine samples from controls containing 01000 nmol/ml of t-amyl alcohol and 2-methyl-2,3-butane diol. The method was linear in the range of concentrations used and calibration standards were analyzed with every sample series (usually 2030 samples). The method permitted the quantitation of 0.1 nmol of t-amyl alcohol and 0.5 nmol 2methyl-2,3-butane diol glucuronide with a signal-to-noise ratio of 3:1. When identical samples were repeatedly analyzed, the coefficients of variation were lower than 10% (n = 8). This method determined the content of free t-amyl alcohol and free 2-methyl-2,3-butane diol and their glucuronides in the samples. Concentrations of the two glucuronides were obtained by subtraction of the content of the free alcohols determined as described below.
To quantify 2-methyl 2,3-butane diol, 25 µl of a solution of the internal standard 1,2-propane diol (1000 nmol/ml in water) was added to 0.1 ml of human or rat urine. Urine samples were then diluted with 0.9 ml methanol, and 2-methyl-2,3-butane diol content was quantified by GC/MS by injecting 1 µl of the obtained mixtures. Separation was achieved using a fused silica column coated with DB-FFAP (30 m x 0.32 mm, film thickness 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 230°C. The concentrations of 2-methyl-2,3-butane diol were determined by monitoring m/z 59 and m/z 45 during the gas chromatographic separation, with dwell times of 80 msec. 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 01000 nmol/ml 2-methyl 2,3-butane diol. The method was linear in the range of concentrations used and calibration standards were analyzed with every sample series (usually 2030 samples) The method permitted the quantitation of 1 nmol 2-methyl 2,3-butane diol/ml of urine with a signal-to-noise ratio of 5:1. When identical samples were repeatedly analyzed, the coefficients of variation were lower than 15% (n = 8).
Concentrations of 2-hydroxy-2-methylbutyric acid and 3-hydroxy-3-methylbutyric acid in urine were quantified by GC/MS after transformation to the corresponding methyl esters. Urine samples (0.1 ml for humans and rats) were mixed with 2-hydroxyvaleric acid (internal standard, 25 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 water and extracted with 1 ml chloroform. The chloroform layers were dried over sodium sulfate and 2 µl of the obtained solutions 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 55 and 59 were monitored during the separation with dwell times of 80 msec. Quantitation was performed relative to the content of 2-hydroxyvaleric acid and referenced to calibration curves with fortified aliquots of urine samples from controls containing 01000 nmol/ml 2-hydroxy-2-methylbutyric acid and 3-hydroxy-3-methylbutyric acid. The method was linear in the range of concentrations used and calibration standards were analyzed with every sample series (usually 2030 samples) The method permitted the quantitation of 3 nmol 2-hydroxy-2-methylbutyric acid and 3-hydroxy-3-methylbutyric /ml of urine with a signal-to-noise ratio of 3:1. When identical samples were repeatedly analyzed, the coefficients of variation were lower than 10% (n = 8).
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). Samples were also analyzed with electron impact ionization using a Hewlett-Packard 5970 mass spectrometer coupled to a 5890 GC or a Hewlett-Packard 5973 mass spectrometer coupled to a 6890 GC. Both instruments were equipped with a CTC Combi-PAL autoinjector with capability for headspace injection.
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 sets from the male and female animals and male and female human volunteers were compared using Student's t-test in Microsoft Excel spreadsheets. p-Values of less than 0.05 were considered significant. Half-lives were calculated using exponential regression in Microsoft ExcelR spreadsheets. The curve-fitting function of the program was used and curves were stripped based on correlation coefficients. r2-Values of > 0.95 were considered for separation.
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RESULTS |
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Due to the absence of background, the concentrations of TAME and t-amyl alcohol could be quantified with higher precision. TAME was detectable in all urine samples from the volunteers collected between 0 and 12 h after the end of the exposure after 40 ppm TAME and between 0 and 6 h after the end of exposure after 4 ppm TAME. t-Amyl alcohol and its glucuronide were also detected in all urine samples collected between 0 and 36 h after the end of the 40-ppm exposure in low concentrations. Excretion of these compounds with urine was rapid and occurred with half-lives of less than 10 h.
Based on the recovered amounts of 2-methyl-2,3-butane diol and 2-hydroxy-2-methylbutyric acid, these compounds represent the major excretory metabolites formed from TAME (Tables 3 and 4) in humans. In addition, 3-hydroxy-3-methylbutyric acid was a TAME metabolite present in higher concentrations in urine, whereas free and conjugated t-amyl alcohol and unchanged TAME were only minor excretory products. Large variations in the extent of TAME biotransformation (Table 4
) between the individuals and in the rates of excretion and the urinary concentrations of 2-hydroxy-2-methylbutyric acid and 3-hydroxy-3-methylbutyric acid were observed. However, no statistically significant differences in the amounts of these acids, of free and conjugated 2-methyl-2,3-butane diol or any of the other metabolites excreted, or in the rates of excretion of metabolites were seen between the male and female subjects in the study. The determined half-lives of elimination with urine were not significantly different after the 4 and 40 ppm TAME exposures (Tables 3 and 4
).
Biotransformation of TAME in Rats
Rats were exposed to the same TAME concentrations as used in the human studies. The experimental results on metabolite concentrations and excretion are compiled in Tables 57 and in Figures 4 and 5
. The concentrations of TAME in blood of rats determined after the end of the 4-h exposure period were twice as high as those seen in humans after identical exposure concentrations. TAME was more rapidly cleared from rat blood as compared to human blood. The concentrations of t-amyl alcohol were not significantly different between rats and humans after 4 and 40 ppm TAME.
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Based on the amounts of 2-methyl-2,3-butane diol and its glucuronide, these compounds and t-amyl alcohol glucuronide represent the major urinary metabolites of TAME (Table 6) in rat urine. Assuming identical retention of TAME after inhalation exposure, humans excreted a significantly higher proportion (p < 0.03) of the retained TAME as metabolites in urine as compared to rats (Tables 5 and 7
).
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DISCUSSION |
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Half-lives of elimination of TAME from blood were also similar to those seen with other ethers intended for use as oxygenates determined in other studies. Elimination of methyl tert-butyl ether and ethyl tert-butyl ether also occurs with half-lives of less than 1 h in rats and (Miller et al., 1997) between 2 and 4 h in humans (Nihlen et al., 1998
; Prah et al., 1994
). As seen with these ethers, no sex differences in the apparent half-lives of elimination of these compounds were seen.
In urine of humans and rats, the known metabolites of TAME excreted with urine and unchanged TAME were quantified. A minor part of the TAME dose is excreted with urine in humans. Elimination of the parent ether with urine was also seen with MTBE and ETBE (Amberg et al., 1999b, 2000
; Nihlen et al., 1998
; Prah et al., 1994
). With these compounds, urinary elimination of the ether also represents a minor pathway for the clearance of the ethers. In rat urine, the TAME concentrations in the urine samples were already below the limit of detection in the first available samples (6 h after the end of exposure).
The study confirms the structure of TAME metabolites formed in the rat and demonstrates that identical metabolites of TAME are formed in humans and excreted with urine. Moreover, the obtained results confirm and extend the semiquantitative findings from our previous study on differences in the metabolic pattern of TAME between humans and rats (Amberg et al., 1999a). In rats, TAME is mainly excreted as 2-methyl-2,3-butane diol and its glucuronide. Further oxidation of t-amyl alcohol to other products is of minor importance due to a more rapid elimination and glucuronide formation. In humans, 2-methyl-2,3-butane diol is eliminated more slowly as compared to rats. In addition, t-amyl alcohol seems to be more efficiently oxidized to 2-hydroxy-2-methylbutyric acid and 3-hydroxy-3-methylbutyric acid in humans. This reaction occurs only to a minor extent in rats. These differences are likely due to substrate specificities of the enzymes involved in the formation of these compounds from TAME and t-amyl alcohol (cytochrome P450 and glucuronyl transferase).
All TAME metabolites quantified in this study were eliminated in rats with apparent half-lives of elimination of less than 6 h; in humans, the elimination with urine of the metabolites formed from TAME was considerably slower.
The extent of TAME biotransformation in humans is significantly higher as compared to rats when the amounts of metabolites and the relative concentrations recovered in urine are compared to the doses received (Tables 5 and 7). Between 40% (rats) and almost 60% (humans) of the calculated doses of TAME received by inhalation were recovered as metabolites in urine. The rest of the TAME taken up by inhalation is likely exhaled. Exhalation of unchanged TAME was not determined in this study. However, due to the volatility of TAME and based on studies with the structurally similar compounds methyl tert-butyl ether and ethyl tert-butyl ether, it has to be assumed that the unaccounted part of the received TAME-dose is exhaled unchanged either by rats or by humans after the termination of the inhalation period. Exhalation of unchanged parent ether was a major pathway of elimination of both methyl tert-butyl ether and ethyl tert-butyl ether in humans and in rats. Extent of biotransformation of TAME in both rats and humans seems not to be saturated in the concentration range studied, as the percentage of dose recovered as metabolites was identical after both exposure concentrations. The interindividual differences in the extent of TAME biotransformation in humans are likely due to interindividual differences in the cytochrome P450 profile (Hong et al., 1999
). More than 10-fold differences in the capacity of human liver microsomes to oxidize TAME to t-amyl alcohol were observed (Hong et al., 1999
). Further studies identifying the cytochrome P450 enzymes involved in t-amyl alcohol biotransformation should address this problem.
The differences in biotransformation and kinetics of metabolite excretion suggest that humans may respond differently than rats to potential toxic effects of TAME based on biotransformation. However, as only limited data on toxic effects of TAME after long-term inhalation exposure in rats are available, conclusions must be taken with caution. The potential of TAME to induce acute and chronic toxicity in rats is low, and the role of biotransformation in the observed changes in relative organ weights is unknown. As the formation of reactive metabolites during TAME biotransformation is not suggested based on the structures of metabolites and their mechanisms of formation, covalent binding to macromolecules has to be regarded as unlikely; thresholds are likely for TAME toxicity. Moreover, the major TAME metabolites found in humans are also formed endogenously (Liebich and Forst, 1984). TAME exposures in low concentrations as expected from the environment are not likely to result in a significant increase in the human body burden of these compounds, making toxic effects of TAME in humans under realistic exposure conditions unlikely.
In conclusion, the biotransformation of TAME after inhalation is quantitatively different, but qualitatively similar in rats and humans. Sex differences in biotransformation were not seen in either species. 2-Methyl-2,3-butane diol concentrations in urine or blood may represent useful biomarkers of exposure to low levels of TAME expected from environmental exposures.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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