Metabolism of methyl tert-butyl ether and other gasoline ethers by human liver microsomes and heterologously expressed human cytochromes P450: identification of CYP2A6 as a major catalyst.

To reduce the production of carbon monoxide and other pollutants in motor vehicle exhaust, methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME) are added to gasoline as oxygenates for more complete combustion. Previously, we demonstrated that human liver is active in metabolizing MTBE to tert-butyl alcohol (TBA) and that cytochrome P450 (CYP) enzymes play a critical role in the metabolism of MTBE. The present study demonstrates that human liver is also active in the oxidative metabolism of ETBE and TAME. A large interindividual variation in metabolizing these gasoline ethers was observed in 15 human liver microsomal samples. The microsomal activities in metabolizing MTBE, ETBE, and TAME were highly correlated among each other (r, 0.91-0. 96), suggesting that these ethers are metabolized by the same enzyme(s). Correlation analysis of the ether-metabolizing activities with individual CYP enzyme activities in the liver microsomes showed that the highest degree of correlation was with human CYP2A6 (r, 0. 90-0.95), which is constitutively expressed in human livers and known to be polymorphic. CYP2A6 displayed the highest turnover number in metabolizing gasoline ethers among a battery of human CYP enzymes expressed in human B-lymphoblastoid cells. Kinetic studies on MTBE metabolism with three human liver microsomes exhibited apparent Km values that ranged from 28 to 89 microM and the V(max) values from 215 to 783 pmol/min/mg, with similar catalytic efficiency values (7.7 to 8.8 microl/min/mg protein). Metabolism of MTBE, ETBE, and TAME by human liver microsomes was inhibited by coumarin, a known substrate of human CYP2A6, in a concentration-dependent manner. Monoclonal antibody against human CYP2A6 caused a significant inhibition (75% to 95%) of the metabolism of MTBE, ETBE, and TAME in human liver microsomes. Taken together, these results clearly indicate that in human liver, CYP2A6 is the major enzyme responsible for the metabolism of MTBE, ETBE, and TAME.     

Human cytochrome P450 2A6 is the major enzyme involved in the metabolism of three alkoxyethers used as oxyfuels.

Methyl t-butyl ether (MTBE), ethyl t-butyl ether (ETBE), and t-amyl methyl ether (TAME) are three alkoxyethers added to gasoline to improve combustion and thereby to reduce the level of carbon monoxide and aromatic hydrocarbons in automobile exhaust. Oxidative demethylation of MTBE and TAME and deethylation of ETBE by CYP enzymes results in the formation of tertiary alcohols and aldehydes, both potentially toxic. The metabolism of these three alkoxyethers was studied in a panel of 12 human liver microsomes. The relatively low apparent Km(1) was 0.25+/-0.17 (mean+/-SD), 0.11+/-0.08 and 0.10+/-0.07 mM and the high apparent Km(2) was 2.9+/-1.8, 5.0+/-2.7 and 1.7+/-1.0 mM for MTBE, ETBE and TAME, respectively. Kinetic data, correlation studies, chemical inhibition and metabolism by heterologously expressed human CYPs support the assertion that the major enzyme involved in MTBE, ETBE and TAME metabolisms is CYP2A6, with a minor contribution of CYP3A4 at low substrate concentration.     

Rat Olfactory Mucosa Displays a High Activity in Metabolizing Methyl tert-butyl Ether and Other Gasoline Ethers

Methyl tert-butyl ether (MTBE) is a widely used gasoline oxygenate.Two other ethers, ethyl tert-butyl ether (ETBE) and tert-amylmethyl ether (TAME), are also used in reformulated gasoline.Inhalation is a major route for human exposure to MTBE and othergasoline ethers. The possible adverse effects of MTBE in humansare a public concern and some of the reported symptoms attributedto MTBE exposure appear to be related to olfactory sensation.In the present study, we have demonstrated that the olfactorymucosa of the male Sprague-Dawley rat possesses the highestmicrosomal activities, among the tissues examined, in metabolizingMTBE, ETBE, and TAME. The metabolic activity of the olfactorymucosa was 46-fold higher than that of the liver in metabolizingMTBE, and 37- and 25-fold higher, respectively, in metabolizingETBE and TAME. No detectable activities were found in the microsomesprepared from the lungs, kidneys, and olfactory bulbs of thebrain. The observations that the metabolic activity was localizedexclusively in the microsomal fraction, depended on the presenceof NADPH, and was inhibitable by carbon monoxide are consistentwith our recent report on MTBE metabolism in human and mouselivers (Hong et al., 1997) and further confirm that cytochromeP450 enzymes play a critical role in the metabolism of MTBE,ETBE, and TAME. The apparent K_m and V_max values for the metabolismof MTBE, ETBE, and TAME in rat olfactory microsomes were verysimilar, ranging from 87 to 125 µM and 9.8 to 11.7 nmol/min/mgprotein, respectively. Addition of TAMIE (0.1 to 0.5 mM) intothe incubation mixture caused a concentration-dependent inhibitionof the metabolism of MTBE and ETBE. Coumarin (50 µM) inhibitedthe metabolism of these ethers by approximately 87%. Furthercomparative studies with human nasal tissues on the metabolismof these ethers are needed in order to assess the human relevanceof our present findings.     

Role of cytochromes P450 in the metabolism of methyl tert -butyl ether in human livers

Methyl tert-butyl ether (MTBE) is widely used as a gasoline oxygenate for more complete combustion in order to reduce the air pollution caused by motor vehicle exhaust. The possible adverse effects of MTBE on human health is a major public concern. However, information on the metabolism of MTBE in human tissues is lacking. The present study demonstrates that human liver is active in metabolizing MTBE to tert-butyl alcohol (TBA), a major circulating metabolite and a marker for exposure to MTBE. The activity is localized in the microsomal fraction (125 ± 11 pmol TBA/min per mg protein, n = 8) but not in the cytosol. This activity level in human liver microsomes is approximately one-half of the value in rat and mouse liver microsomes. Formation of TBA in human liver microsomes is NADPH-dependent, and is significantly inhibited by carbon monoxide (CO), an inhibitor of cytochrome P450 (CYP) enzymes, suggesting that CYP enzymes play a critical role in the metabolism of MTBE in human livers. Both CYP2A6 and 2E1 are known to be constitutively expressed in human livers. To examine their involvement in MTBE metabolism, human CYP2A6 and 2E1 cDNAs were individually co-expressed with human cytochrome P450 reductase by a baculovirus expression system and the expressed enzymes were used for MTBE metabolism. The turnover number for CYP2A6 and 2E1 was 6.1 and 0.7 nmol TBA/min per nmol P450, respectively. The heterologously expressed human CYP2A6 was also more active than 2E1 in the metabolism of two other gasoline ethers, ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether (TAME). Although the contributions of other human CYP forms to MTBE metabolism remain to be determined, these results strongly suggest that CYP enzymes play an important role in the metabolism of MTBE in human livers. Received: 2 July 1996 / Accepted: 7 November 1996     

Interactive effects of methyl tertiary-butyl ether (MTBE) and tertiary-amyl methyl ether (TAME), ethanol and some drugs: Triglyceridemia, liver toxicity and induction of CYP (2E1, 2B1) and phase II enzymes in female Wistar rats

The abilities of the gasoline additives methyl tert-butyl ether (MTBE) and tert-amyl methyl ether (TAME) to cause liver damage following oral administration, dosed alone or in combination with model hepatotoxins, were investigated in the rat. Inducibility of liver drug-metabolizing enzyme activities was also studied. Exposure to these ethers (10-20mmol/kg) for 3 days resulted in hepatomegaly (13-30%) and induction of cytochrome P450 (CYP) activity towards N-nitrosodimethylamine (NDMAD), 7-pentoxyresorufin (PROD), and 7-ethoxyresorufin (EROD). Immunoinhibition assays with monoclonal antibodies showed that the ethers were equipotent as inducers of CYP2E1 activity (2-fold increase) but not of CYP2B1, which was elevated up to 260-fold in TAME-treated rats but only by 20-fold in MTBE rats. A slight or no modifying effect was observed on the NADPH:quinone oxidoreductase (NQO1), glutathione S-transferase (GST), and UDP-glucuronosyltransferase (UGT) activities. Alanine aminotransaminase (ALT) and aspartate aminotransaminase (AST) were elevated in blood plasma after administration of the ethers. No dramatic enhancement of liver damage could be detected by plasma enzyme analysis (ALT, AST, alkaline phosphatase, γ-glutamyltransferase) following ether administration (13.5mmol/kg) to rats pretreated with mildly hepatotoxic dosages of ethanol, pyrazole, phenobarbital, acetaminophen (paracetamol), or 13-cis-retinoic acid (13-cis-RA or isotretinoin). Plasma triglycerides increased in TAME-treated rats (1.7-fold) and in all 13-cis-RA-treated groups (2.1-2.8-fold). The findings that MTBE and TAME exhibited a clear but differential inducing effect on two ether-metabolizing CYP forms (2E1 and 2B1) with no marked effect on phase II activities may reflect the importance of these pathways in vivo. The observation that only TAME by itself induced hypertriglyceridemia while acetaminophen- and 13-cis-RA-induced hypertriglyceridemia were aggravated by both ethers, points to differences in their effects on lipid metabolism. TAME was clearly a more potent CNS depressant than MTBE. There was no marked potentiation of drug/chemical-induced acute liver damage either by MTBE or TAME.     

Oxidation of methyl- and ethyl- tertiary-butyl ethers in rat liver microsomes: role of the cytochrome P450 isoforms

Methyl t-butyl ether (MTBE) and ethyl t-butyl ether (ETBE) are commonly used in unleaded gasoline to increase the oxygen content of fuel and to reduce carbon monoxide emissions from motor vehicles. This study was undertaken to investigate: (1) the effect of administration to rats of ETBE and its metabolite, t-butanol, on the induction and/or inhibition of hepatic P450 isoenzymes; (2) the oxidative metabolism of MTBE and ETBE by liver microsomes from rats pretreated with selected P450 inducers and purified rat P450(s), (2B1, 2E1, 2C11, 1A1). ETBE administration by gavage at a dose of 2 ml/kg for 2 days induced hepatic microsomal P4502E1-linked p-nitrophenol hydroxylase and the P4502B1/2-associated PROD and 16β-testosterone hydroxylase, verified by immunoblot experiments. t-Butanol treatments at doses of 200 and 400 mg/kg i. p. for 4 days did not alter any liver microsomal monoxygenases. Both MTBE and ETBE were substrates for rat liver microsomes and were oxidatively dealkylated to yield formaldehyde and acetaldehyde, respectively. The dealkylation rates of both MTBE and ETBE were increased c. fourfold in phenobarbital (PB)-treated rats. In rats pretreated with pyrazole, an inducer of 2E1, only the demethylation of MTBE was increased (c. twofold). When the oxidations of MTBE and ETBE were investigated with purified P450(s) in a reconstituted system, it was found that P4502B1 had the highest activities towards both solvents, whereas 1A1 and 2C11 were only slightly active; P4502E1 had an appreciable activity on MTBE but not against ETBE. Metyrapone, a potent inhibitor of P450 2B, consistently inhibited both the MTBE and ETBE dealkylations in microsomes from PB-treated rats. Furthermore, 4-methylpyrazole (a probe inhibitor of 2E1) and anti-P4502E1 IgG showed inhibition, though modest, only on MTBE demethylation, but not on ETBE deethylation. Inhibition experiments have also suggested that rat 2A1 may exert an important role in MTBE and ETBE oxidation. Taken together, these results indicate that 2B1, when expressed, is the major enzyme involved in the oxidation of these two solvents and that 2E1 may have a role, although minor, in MTBE demethylation. The implications of these data for MTBE and ETBE toxicity remain to be established. Received: 5 August 1997 / Accepted: 29 October 1997     

Toxicokinetics of ethers used as fuel oxygenates.

The toxicokinetics and biotransformation of methyl-tert.butyl ether (MTBE), ethyl-tert.butyl ether (ETBE) and tert.amyl-methyl ether (TAME) in rats and humans are summarized. These ethers are used as gasoline additives in large amounts, and thus, a considerable potential for human exposure exists. After inhalation exposure MTBE, ETBE and TAME are rapidly taken up by both rats and humans; after termination of exposure, clearance by exhalation and biotransformation to urinary metabolites is rapid in rats. In humans, clearance by exhalation is slower in comparison to rats. Biotransformation of MTBE and ETBE is both qualitatively and quantitatively similar in humans and rats after inhalation exposure under identical conditions. The extent of biotransformation of TAME is also quantitatively similar in rats and humans; the metabolic pathways, however, are different. The results suggest that reactive and potentially toxic metabolites are not formed during biotransformation of these ethers and that toxic effects of these compounds initiated by covalent binding to cellular macromolecules are unlikely.     

Quantification of fuel oxygenate ethers in human blood using solid-phase microextraction coupled with gas chromatography-high-resolution mass spectrometry

Widespread use of fuel oxygenates, coupled with their high water solubility and slow degradation rate, have led to an increase in the potential for human exposure. We developed an accurate, precise, sensitive, and high-throughput analytical method to simultaneously quantify trace levels (low parts-per-trillion) of four fuel oxygenates in human blood: methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), di-isopropyl ether (DIPE), and tert-amyl methyl ether (TAME). The analytes were extracted from the head space above human blood samples, using solid-phase microextraction, desorbed into the heated injector, and chromatographically resolved by capillary gas chromatography. Analytes were detected by high-resolution mass spectrometry with multiple ion monitoring, and quantified against known standard levels by use of stable isotope-labeled internal standards for recovery correction. The low limits of detection (0.6 ng/L) allowed for measurement of MTBE, ETBE, DIPE, and TAME in parts-per-trillion levels with excellent precision (coefficient of variation ranging from 1.7 to 5.4%) and accuracy (96-100%). This method provides a means to assess fuel oxygenate exposure and study the potential relationship between exposure and adverse health outcomes.     

Toxicology and human health effects following exposure to oxygenated or reformulated gasoline

In order to replace antiknock leaded derivatives in gasoline, legislations were enacted in the United States and other countries to find safer additives and to reduce CO, O3, and volatile organic compounds (VOCs) in non-attainment areas. Oxygenates commonly used include various alcohols and aliphatic ethers. Methyl tert-butyl ether (MTBE) is the most widely used and studied ether oxygenate and is added to gasoline at concentrations up to 15% by volume. Inhalation of fumes while fueling automobiles is the main source of human exposure to MTBE. Humans are also exposed when drinking water contaminated with MTBE. Epidemiological, clinical, animal, metabolic and kinetic studies have been carried out to address human health risks resulting from exposure to MTBE. MTBE is an animal carcinogen, but its human carcinogenic potential remains unclear. Because MTBE functions as a non-traditional genotoxicant, several mechanisms were suggested to explain its mode of action, such as, functioning as a cytotoxic as opposed to a mitogenic agent; involvement of hormonal mechanisms; or operating as a promoter instead of being a complete carcinogen. Some studies suggested that carcinogenicity of MTBE might be due to its two main metabolites, formaldehyde or tributanol. A role for DNA repair in MTBE carcinogenesis was recently unveiled, which explains some, but not all effects. The totality of the evidence shows that, for the majority of the non-occupationally exposed human population, MTBE is unlikely to produce lasting adverse health effects, and may in some cases improve health by reducing the composition of emitted harmful VOCs and other substances. A small segment of the population (e.g. asthmatic children, the elderly, and those with immunodeficiency) may be at increased risk for toxicity. However, no studies have been conducted to investigate this hypothesis. Concern over ground and surface water contamination caused by persistent MTBE has lead the Environmental Protection Agency (EPA) to proposed reducing or eliminating its use as a gasoline additive. The major potential alternatives to MTBE are other forms of ethers such as ethyl tert-butyl ether (ETBE) or tert-amyl methyl ether (TAME), and alcohols such as ethanol. More definitive studies are needed to understand the mechanism(s) by which aliphatic ethers may pose health and environmental impacts. The switch from MTBE to ethanol is not without problems. Ethanol costs more to produce, poses challenges to the gasoline distribution system, extends the spread of hydrocarbons through ground water in gasoline plumes, and in the short-term is unlikely to be available in sufficient quantity. Moreover, its metabolite acetaldehyde is a possible carcinogen that undergoes a photochemical reaction in the atmosphere to produce the respiratory irritant peroxylacetate nitrate (PAN). Congress is addressing whether the Clean Air Act Amendments (CAA) provisions concerning reformulated gasoline (RFG) should be modified to allow refineries to discontinue or lessen the use of oxygenates.     

Influence of oxygenated fuel additives and their metabolites on gamma-aminobutyric acidA (GABAA) receptor function in rat brain synaptoneurosomes

Experimental and occupational inhalational exposure to oxygenate fuel additives in reformulated gasoline has been reported to induce neurological symptoms (e.g., headache, nausea, dizziness). We reported previously that the ether additives (methyl-t-butyl ether (MTBE), t-amyl-methyl ether (TAME) and ethyl-t-butyl ether (ETBE)) and their metabolites (t-amyl alcohol (TAA), t-butyl alcohol (TBA) and ethanol) alter the binding of [3H]t-butylbicycloorthobenzoate ([3H]TBOB), a ligand for the gamma-aminobutyric acidA (GABAA) receptor in rat brain membrane preparations. To more directly assess the effects of the ethers and their alcohol precursors on GABAA receptor function, the uptake of 36Cl- was measured in synaptoneurosomes, a preparation of closed membrane sacs comprised of pre- and postsynaptic membranes from adult rat cerebral cortex. Each of the compounds caused a concentration-dependent enhancement of muscimol-stimulated uptake of 36CI-, which diminished with further increasing concentrations. The potency of the enhancement by the compounds was in the rank order: MTBE = TAME > TAA = ETBE > TBA > ethanol. The half-maximally effective concentration (EC50) for the facilitation of muscimol-stimulated 36Cl- uptake ranged from 0.06 to 3 mM, and that for the higher-dose inhibitory effect (IC50) ranged from 3 to 50 mM. The facilitatory concentrations of the compounds are in the range of the blood concentrations reported in experimental animals after exposures known to induce CNS effects such as ataxia. The results suggest a potential role of the GABAA receptor in some of the reported neurotoxic effects of gasoline additives.     

Ether oxygenate additives in gasoline reduce toxicity of exhausts

Fuel additives can improve combustion and knock resistance of gasoline engines. Common additives in commercial fuels are “short-chain, oxygen containing hydrocarbons” such as methyl tert-butyl ether (MTBE) and ethyl tert-butyl ether (ETBE). Since these additives change the combustion characteristics, this may as well influence toxic effects of the resulting emissions. Therefore we compared toxicity and BTEX emissions of gasoline engine exhaust regarding addition of MTBE or ETBE.     

Adduction of DNA with MTBE and TBA in mice studied by accelerator mass spectrometry

Methyl tert-butyl ether (MTBE) is a currently worldwide used octane enhancer substituting for lead alkyls and gasoline oxygenate. Our previous study using doubly (14)C-labeled MTBE [(CH(3))(3) (14)CO(14)CH(3)] has shown that MTBE binds DNA to form DNA adducts at low dose levels in mice. To elucidate the mechanism of the binding reaction, in this study, the DNA adducts with singly (14)C-labeled MTBE, which was synthesized from (14)C-methanol and tert-butyl alcohol (TBA), or (14)C-labeled TBA in mice have been measured by ultra sensitive accelerator mass spectrometry. The results show that the methyl group of MTBE and tert-butyl alcohol definitely form adducts with DNA in mouse liver, lung, and kidney. The methyl group of MTBE is the predominant binding part in liver, while the methyl group and the tert-butyl group give comparable contributions to the adduct formation in lung and kidney.     

Diversity of selective environmental substrates for human cytochrome P450 2A6: alkoxyethers,nicotine, coumarin,N-nitrosodiethylamine,and N-nitrosobenzylmethylamine

Cytochrome P450 2A6 constitutes 5-10% of the total microsomal CYPs of human liver. Although CYP2A6 is the major coumarin 7-hydroxylase, other known substrates of CYP2A6 include many toxicants and precarcinogens. The chemical structure diversity of these substrates raises the question of their selectivity. Thus, kinetic parameters were determined for the hydroxylation of five substrates of diverse chemical structures known to be selective for cytochrome P450 2A6: methyl tert-butyl ether (MTBE), nicotine, coumarin, N-nitrosobenzylmethylamine (NBzMA), and N-nitrosodiethylamine (NDEA). Sources of enzymes were either human liver microsomes or heterologously expressed CYPs. Coumarin was shown to be the substrate with the highest affinity, followed by NDEA, nicotine, NBzMA, and MTBE. Variability of CYP2A6 catalytic activities in human liver was between 24-fold for MTBE to sevenfold for coumarin, while CYP2A6 content varied 68-fold in human liver microsomes. These five catalytic activities were highly significantly correlated between them and with hepatic CYP2A6 content. The most selective chemical inhibitor of these five substrates was shown to be 8-methoxypsoralen. Based upon chemical inhibition of the enzymatic activities of pure recombinant human CYPs, it cannot be totally excluded that P450s other than CYP2A6, especially CYP2E1, are involved, although to a lesser extent, in NDEA and NBzMA metabolism. In conclusion, the prototype probes for CYP2A6 phenotyping are coumarin and nicotine.     

In vivo exposure of female rats to toxicants may affect oocyte quality

A potential endpoint for female reproductive toxicants is fertilizability of the oocytes. This endpoint has not been adequately examined for mammalian females. The objective of these studies was to evaluate fertilizability of rat oocytes following in vivo exposure to known male reproductive toxicants that exert effects via pathways that do not include endocrine disruption and to 4-vinylcyclohexene diepoxide, known to interfere with early follicular development. Oocytes were obtained from females following exposure and quality assessed by in vitro fertilization rate. One study evaluated fertilizability following 2 weeks exposure of females to inhaled tetrachloroethylene (2h/day, 5 days/week). The remaining studies evaluated fertilizability immediately following 2 weeks exposure via drinking water to tetrachloroethylene, trichloroethylene, the fuel oxidants methyl tertiary butyl ether (MTBE), ethyl tertiary butyl ether (ETBE), tertiary amyl methyl ether (TAME), and a metabolite of the first two ethers 2-methyl-1,2-propanediol (2M2P), and to 4-vinylcyclohexene diepoxide. The percentage of oocytes fertilized was reduced following inhalation exposure to tetrachloroethylene, or consumption of trichloroethylene or TAME. Fertilizability was not altered by exposures to the other reproductive toxicants or to the other fuel oxidants. Consistent with the reduced oocyte fertilizability following exposure to trichloroethylene, oocytes from exposed females had a reduced ability to bind sperm plasma membrane proteins. Female reproductive capability assessed by the endpoint, oocyte fertilizability, was reduced by exposure to trichloroethylene and inhaled tetrachloroethylene.     

Effects of a thirteen-week inhalation exposure to ethyl tertiary butyl ether on fischer-344 rats and CD-1 mice.

The 1990 Clean Air Act Amendments require that oxygenates be added to automotive fuels to reduce emissions of carbon monoxide and hydrocarbons. One potential oxygenate is the aliphatic ether ethyl tertiary butyl ether (ETBE). Our objective was to provide data on the potential toxic effects of ETBE. Male and female Fisher 344 rats and CD-1 mice were exposed to 0 (control), 500, 1750, or 5000 ppm of ETBE for 6 h/day and 5 days/wk over a 13-week period. ETBE exposure had no effect on mortality and body weight with the exception of an increase in body weights of the female rats in the 5000-ppm group. No major changes in clinical pathology parameters were noted for either rats or mice exposed to ETBE for 6 (rats only) or 13 weeks. Liver weights increased with increasing ETBE-exposure concentration for both sexes of rats and mice. Increases in kidney, adrenal, and heart (females only) weights were noted in rats. Degenerative changes in testicular seminiferous tubules were observed in male rats exposed to 1750 and 5000 ppm but were not seen in mice. This testicular lesion has not been reported previously for aliphatic ethers. Increases in the incidence of regenerative foci, rates of renal cell proliferation, and alpha2u-globulin containing protein droplets were noted in the kidneys of all treated male rats. These lesions are associated with the male rat-specific syndrome of alpha2u-globulin nephropathy. Increases in the incidence of centrilobular hepatocyte hypertrophy and rates of hepatocyte cell proliferation were seen in the livers of male and female mice in the 5000-ppm group, consistent with a mitogenic response to ETBE. These two target organs for ETBE toxicity, mouse liver and male rat kidney, have also been reported for methyl tertiary butyl ether and unleaded gasoline.     

Species and gender differences in the metabolism and distribution of tertiary amyl methyl ether in male and female rats and mice after inhalation exposure or gavage administration

Tertiary amyl methyl ether (TAME) is a gasoline fuel additive used to reduce emissions. Understanding the metabolism and distribution of TAME is needed to assess potential human health issues. The effect of dose level, duration of exposure and route of administration on the metabolism and distribution of TAME were investigated in male and female F344 rats and CD-1 mice following inhalation or gavage administration. By 48 h after exposure, >96% of the administered radioactivity was expired in air (16-71%) or eliminated in urine and feces (28-72%). Following inhalation exposure, mice had a two- to threefold greater relative uptake of [14C]TAME compared with rats. Metabolites were excreted in urine of rats and mice that are formed by glucuronide conjugation of tertiary amyl alcohol (TAA), oxidation of TAA to 2,3-dihydroxy-2-methylbutane and glucuronide conjugation of 2,3-dihydroxy-2-methylbutane. A saturation in the uptake and metabolism of TAME with increased exposure concentration was indicated by a decreased relative uptake of total [14C]TAME equivalents and an increase in the percentage expired as volatiles. A saturation of P-450 oxidation of TAA was indicated by a disproportional decrease of 2,3-dihydroxy-2-methylbutane and its glucuronide conjugate with increased exposure concentration.     

Influence of oxygenated fuel additives and their metabolites on the binding of a convulsant ligand of the gamma-aminobutyric acid(A) (GABA(A)) receptor in rat brain membrane preparations

As a foundation for evaluating potential mechanisms of the neurological effects (e.g. headache, nausea, dizziness) of some octane boosters, we studied the gamma-aminobutyric acid(A) (GABA(A)) receptor in a series of binding assays in membranes from rat brain. The GABA(A) receptor was probed using the radioligand [3H]t-butylbicycloorthobenzoate ([3H]TBOB) which binds to the convulsant recognition site of the receptor. The results demonstrated that the short-chain t-ethers and their t-alcohol metabolites inhibit binding at the convulsant site of the GABA(A) receptor. The potency of the inhibition tended to correlate with carbon chain length. For agents having an equal number of carbon atoms, potency of inhibition of [3H]TBOB binding was greater in magnitude for the alcohols than for the ethers. The descending rank order of potency for the ethers and alcohols were as follows, t-amyl alcohol (TAA); t-amyl-methyl ether (TAME); ethyl-t-butyl ether (ETBE)>t-butyl alcohol (TBA)>methyl-t-butyl ether (MTBE)>ethanol. In additional saturation binding assays, MTBE reduced apparent density of convulsant binding (B(max)).     

Induction of testosterone biotransformation enzymes following oral administration of methyl tert-butyl ether to male Sprague-Dawley rats.

Methyl tert-butyl ether (MTBE) is an oxygenated fuel additive used to decrease carbon monoxide emissions during gasoline combustion. In the current study, we investigated the hypothesis that the MTBE-induced decrease in serum testosterone levels in male rats may be due in part to the ability of MTBE to induce the metabolism of endogenous testosterone and hence enhance its clearance. Nine-week-old male Sprague-Dawley rats were gavaged with 250, 500, 1000, or 1500 mg MTBE/kg/day in corn oil or corn oil alone for 15 or 28 consecutive days. Increased relative liver weight (10-14%) and minimal-to-moderate centrilobular hypertrophy were observed in rats treated with 1000 and 1500 mg MTBE/kg/day (high doses) for 28 days. Total hepatic microsomal cytochrome P450 (CYP) was increased 1. 3-fold in the high-dose, 15-day-treated rats. An evaluation of specific CYP activities using selective markers demonstrated a 2. 0-fold increase in CYP2B1/2 in rats treated with 1000 mg MTBE/kg/day for 28 days, and with 1500 mg MTBE/kg/day for 15 and 28 days (6.5- and 2.9-fold, respectively). CYP1A1/2, CYP2A1, and CYP2E1 activities were increased 1.5-, 2.4-, and 2.3-fold, respectively, in high-dose, 15-day-treated rats. CYP2E1 was also increased in high-dose, 28-day-treated rats (2.0-fold). CYP3A1/2 was increased 2.1-fold and UDP-glucuronosyltransferase activity 1.7-fold in high-dose, 28-day-treated rats. MTBE also induced its own metabolism 2.1-fold in high-dose, 28-day-treated rats. Results indicate that MTBE induces selected enzymes involved in testosterone metabolism. The decrease in serum testosterone observed following MTBE administration may be the result of enhanced testosterone metabolism and subsequent clearance.     

Blood pharmacokinetics of tertiary amyl methyl ether in male and female F344 rats and CD-1 mice after nose-only inhalation exposure

Interest in understanding the biological behavior of aliphatic ethers has increased owing to their use as gasoline additives. The purpose of this study was to investigate the blood pharmacokinetics of the oxygenate tertiary amyl methyl ether (TAME), its major metabolite tertiary amyl alcohol (TAA) and acetone in rats and mice following inhalation exposure to TAME. Species differences in the area under the curve (AUC) for TAME were significant at each exposure concentration. For rats, the blood TAME AUC increased in proportion with an increase in exposure concentration. For mice, an increase in exposure concentration (100-500 ppm) resulted in a disproportional increase in the TAME AUC. Mice had greater (two- to threefold) blood concentrations of TAA compared with rats following exposure to 2500 or 500 ppm TAME. Mice had a disproportional increase in the TAA AUC with an increase in exposure concentration (100-500 ppm). This difference could result from saturation of a process (e.g. oxidation, glucuronide conjugation) that is involved in the further metabolism of TAA. For each species, gender and exposure concentration, acetone increased during exposure and returned to control values by 16 h following exposure. The source of acetone could be both as a metabolite of TAA or an effect on endogenous metabolism produced by exposure to TAME.     

Controlled Ethyl tert-Butyl Ether (ETBE) Exposure of Male Volunteers: I. Toxicokinetics

Ethyl tert-butyl ether (ETBE) might replace methyl tert-butylether (MTBE), a widely used additive in unleaded gasoline. Theaim of this study was to evaluate uptake and disposition ofETBE, and eight healthy male volunteers were exposed to ETBEvapor (0, 5, 25, and 50 ppm) during 2 h of light physical exercise.ETBE and the proposed metabolites tert-butyl alcohol (TBA) andacetone were analyzed in exhaled air, blood, and urine. Comparedto a previous MTBE study (A. Nihlén et al., 1998b, Toxicol.Appl. Pharmacol. 148, 274–280) lower respiratory uptakeof ETBE (32–34%) was seen as well as a slightly higherrespiratory exhalation (45–50% of absorbed ETBE). Thekinetic profile of ETBE could be described by four phases inblood (average half-times of 2 min, 18 min, 1.7 h, and 28 h)and two phases in urine (8 min and 8.6 h). Postexposure half-timesof TBA in blood and urine were on average 12 and 8 h, respectively.The 48-h pulmonary excretion of TBA accounted for 1.4–3.8%of the absorbed ETBE, on an equimolar basis. Urinary excretionof ETBE and TBA was low, below 1% of the ETBE uptake, indicatingfurther metabolism of TBA or other routes of metabolism andelimination. The kinetics of ETBE and TBA were linear up to50 ppm. Based upon blood profile, levels in blood and urine,and kinetic profile we suggest that TBA is a more appropriatebiomarker for ETBE than the parent ether itself. The acetonelevel in blood was higher after ETBE exposures compared to controlexposure, and acetone is probably partly formed from ETBE.