Chemical Industry Institute of Toxicology, 6 Davis Drive, Research Triangle Park, North Carolina 27709
Received February 3, 2000; accepted June 5, 2000
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ABSTRACT |
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Key Words: methyl tert-butyl ether (MTBE); cytochromes P450; UDPGT; rat liver microsomes; testosterone.
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INTRODUCTION |
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MTBE, along with other gasoline ethers such as ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether (TAME), are metabolized by cytochrome P450 (CYP) enzymes (Hong et al., 1997a,b
). MTBE is metabolized to tert-butyl alcohol and formaldehyde (Brady et al., 1990
) in rodents, primarily by CYP2B1 (Turini et al., 1998
), while CYP2A6 is responsible for MTBE metabolism in humans (Hong et al., 1997b
, 1999a
). Previous investigations into the effects of MTBE on hepatic biotransformation reported a variety of metabolic alterations. Researchers demonstrated an increase in hepatic microsomal CYP activity, hepatic labeling index, and 17ß-estradiol metabolism in female B6C3F1 mice following inhalation exposure to 8000 ppm MTBE (Moser et al., 1998
; 1996a
; 1996b
). These responses may potentially alter the metabolic profile of both exogenous and endogenous compounds. Endogenous steroid hormones, such as testosterone and estradiol, are metabolized in the liver by CYP enzymes to monohydroxylated products, which are further conjugated to polar end products and excreted. In addition, direct conjugation of testosterone by phase-II metabolizing enzymes, namely UDP-glucuronosyltransferases and sulfotransferases, may facilitate the clearance of endogenous testosterone (Griffin and Wilson, 1998
).
A previous study in our laboratory demonstrated a decrease in serum testosterone (52% of control) in Sprague-Dawley rats dosed by gavage with 1500 mg MTBE/kg for 15 days (Williams et al., 2000). This decrease was consistent with a reported decrease in plasma testosterone in Sprague-Dawley rats dosed by gavage with 800 mg MTBE/kg for 28 days (Day et al., 1998
). MTBE-induced alterations in the expression of CYP enzymes may lead to changes in testosterone metabolism and clearance and possibly play a role in the depletion of serum testosterone levels observed in rats. Altered CYP enzyme activity has been associated with a decrease in serum testosterone in cisplatin-treated rats (LeBlanc and Waxman, 1988
) and increased androgen clearance in endosulfan-treated mice (Wilson and LeBlanc, 1998
). In addition, an analogous mechanism has been used for the explanation of thyroid tumors in long-term toxicity studies. Clearance of thyroid hormones is enhanced by an increase in glucuronyltransferase activity, which results in thyroid hypertrophy and eventual tumor formation (Parkinson, 1996
). 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.
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MATERIALS AND METHODS |
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Animals.
Seven-week-old male Sprague-Dawley rats were obtained from Charles River Laboratories (Raleigh, NC) and allowed to acclimate for 14 days prior to use. Rats were randomized by weight (n = 15/dose group) and housed individually in stainless steel and glass 1-m3 Hazelton H1000 inhalation chambers (Lab Products, Inc., Maywood, NJ). Since MTBE has a high vapor pressure and is readily exhaled, one H1000 chamber was used for each dose level of MTBE, to prevent any possible cross-contamination of treated groups that could occur from expired MTBE. Total chamber airflow through each H1000 chamber was maintained at approximately 225 l/min (1215 air changes/h) with the chamber temperature and relative humidity controlled to 23 ± 1°C and 3070%, respectively. All environmental parameters were recorded every 30 min for a 24-h period by an Infinity Building Automation System (Andover Controls Corporation, Andover, MA). Rats had available pelleted food (NIH-07, Zeigler Bros., Gardners, PA) and deionized, filter-purified water ad libitum. A 12-h light-dark cycle was maintained. These studies were performed under National Research Council guidelines (1996) and were approved by the Institutional Animal Care and Use Committee of the Chemical Industry Institute of Toxicology (CIIT).
Study design.
Rats were gavaged with 0 or 1500 mg MTBE/kg/day for 15 consecutive days or 0, 250, 500, 1000, or 1500 mg MTBE/kg/day for 28 consecutive days. The vehicle was corn oil. Dosing solution concentrations were verified using a gas chromatograph with a flame ionization detector. MTBE doses were selected based on previous oral toxicity studies, including a 28-day study in Fischer 344 rats (IIT Research Institute, 1992), a 14- and 90-day study in Sprague-Dawley rats (Robinson et al., 1990
), and a chronic study in Sprague-Dawley rats (Belpoggi et al., 1995
). A previous study in Sprague-Dawley rats reported a decrease in plasma testosterone after administration of 800 mg MTBE/kg for 28 days (Day et al., 1998
). Rats were euthanized by decapitation 1 h following the last dose of MTBE. The livers were removed and weighed. A portion of each liver was fixed in 10% neutral buffered formalin and prepared for H&E staining. The remaining liver was frozen in liquid nitrogen and stored at 80°C. Frozen liver from MTBE-treated male Sprague-Dawley rats was used in this study (Williams et al., 2000
).
Preparation of liver microsomes.
Microsomes were prepared from thawed liver tissue using differential centrifugation according to the method described by Guengerich (1989). The resultant microsomal pellet was suspended in 0.1 M potassium phosphate (pH 7.4) buffer with 0.25 M sucrose and stored at 80°C until use. Microsomal protein concentrations were determined using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL), based on the method of Smith et al. (1985) and using bovine serum albumin as a standard. Total CYP concentrations were measured based on the method of Omura and Sato (1964). Briefly, the reduced-CO and reduced difference spectrum were compared for 1 mg/ml liver microsomes. Spectra were monitored from 400 to 500 nm using a Beckman DU 650 spectrophotometer (Beckman Instruments, Fullerton, CA).
7-Ethoxyresorufin-O-deethylase (EROD) and 7-pentoxyresorufin-O-depentylase (PROD) activities.
EROD and PROD activities were measured using liver microsomes, according to the methods of Prough et al. (1978) and Lubet et al. (1985), respectively. The assay mixtures contained 0.34 mM EROD or 0.20 mM PROD, 25 mM NADPH, 0.2 mg microsomal protein, and 0.05 M Tris-0.025 M MgCl2 buffer (pH 7.4). EROD and PROD activities were measured fluorimetrically by the formation of resorufin periodically over 5 min, using a Perkin-Elmer LS-5 fluorescence spectrophotometer (Norwalk, CT). Resorufin formation was quantitated from a resorufin standard curve.
Testosterone hydroxylase activities.
The microsomal incubations and analyses were performed according to Purdon and Lehman-McKeeman (1997) with minor modifications. Briefly, the incubation mixture contained 0.25 mM testosterone, 1.0 mM NADP, 5.0 mM glucose-6-phosphate, 3 mM MgCl2, 1.0 mM EDTA, 1 U glucose-6-phosphate dehydrogenase, and 0.2 mg microsomal protein. The total incubation volume was 1.0 ml. In control incubation mixtures, testosterone solution was replaced with an equivalent volume of H2O. Incubations were performed for 10 min in a shaking water bath at 37°C. The reactions were terminated with the addition of methylene chloride, and then 1.0 nmol cortexolone was added as an internal standard. The samples were extracted, evaporated, and reconstituted in 150 µl water/methanol (10:1). A 100-µl aliquot of the reconstituted extract was injection into the HPLC system. All incubations were performed in duplicate for each animal.
Analyses of sample extracts were performed on a Hewlett Packard HP1100 Series HPLC system (Avondale, PA) interfaced to a diode array UV/visible detector. A Supelcosil LC-18 column was used (150 x 4.6 mm, 3-µm particle size; Supelco, Bellefonte, PA). Solvent A was house-purified, HPLC-grade water, solvent B was HPLC-grade methanol (Fisher Scientific, Fair Lawn, NJ). Compounds were eluted by a solvent gradient consisting of 10% B to 84% B in 50 min at 1.0 ml/min. The column was flushed with 100% B for 5 min and equilibrated to 10% B for 6 min, resulting in a total analysis time of 61 min. Absorbance was measured at 247 nm. Data analysis was performed using Hewlett-Packard ChemStation 6.01 software (Avondale, PA). Experimental errors were corrected by using the internal standard, cortexolone. The biotransformation products 2-, 2ß-, 6ß-, 7
-, and 16
-hydroxylated testosterones (-OHT) and androstenedione, were quantitated using a standard curve for each metabolite. These metabolic products are noted on the structure of testosterone (Fig. 1
).
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UDP-glucuronosyltransferase (UDPGT) activity.
UDPGT activity was measured using a spectrophotometric assay, according to Bock et al. (1983). Incubation mixtures, which contained 0.1 M TrisHCl (pH 7.4), 5 mM MgCl2, 0.5 mM PNP, and 1 mg/ml liver microsomal protein in 0.25% Triton X-100, were preincubated for 2 min at 37°C prior to the addition of 0.3 mM uridine 5`-diphosphoglucuronic acid (UDPGA). In control incubation mixtures, UDPGA was replaced with an equivalent amount of deionized H2O. The total incubation volume was 0.5 ml. The incubation was performed for 10 min at 37°C in a shaking water bath and terminated by adding 1 ml 5% trichloroacetic acid to 100 µl of the incubation mixture. The sample was diluted 4:1 with 2 M NaOH and the absorbance was measured at 405 nm on a Beckman DU 650 spectrophotometer (Fullerton, CA). The amount of unconjugated 4-nitrophenol remaining was used to determine the extent of glucuronidation using an extinction coefficient of 18.1 cm2/mol (Bock et al., 1983).
MTBE metabolism.
A spectrophotometric method was used to measure formaldehyde formed to evaluate the metabolism of MTBE (Quesenberry and Lee, 1996). Briefly, 1.0 mg microsomal protein in 0.2 M potassium phosphate buffer (pH 7.4) was preincubated for 3 min at 37°C, and then 1.0 mM NADPH and 0.5 µM MTBE were added to a sealed flask. The total incubation volume was 1 ml. The incubation mixture was heated for 10 min at 37°C while shaking. The incubation mixture was diluted 1:1 with 34 mM purpald to terminate the reaction. After 20 min at 25°C, 500 µl of 33 mM NaIO4 was added, and the mixture was allowed to sit at 25°C for 1 h. The absorbance of the mixture was monitored at 550 nm on a Beckman DU 650 spectrophotometer (Fullerton, CA), and formaldehyde production was quantitated using a standard curve. Analyses were performed in duplicate.
Statistical analysis.
Body weights, liver weights, and enzyme activities were analyzed by either a 1-way analysis of variance (ANOVA) or a Student's t-test. Dunnett's test was used to determine significant differences between treatment and control. Trend analyses were performed using Jonckheere's trend test (Jonckheere, 1954). The significance level for all analyses was 0.05.
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RESULTS |
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Hepatic microsomes were evaluated for total microsomal CYP levels along with individual CYP activities using specific markers. A summary of the assays used to measure individual CYP activities is presented in Table 1. Specific CYP activities were selected for measurement, based on their role in testosterone metabolism, single hydroxylation, as well as their role in MTBE metabolism (CYP2B1 and 2E1). Total CYP concentration was significantly higher in rats (n = 1115) treated for 15 days with 1500 mg MTBE/kg/day (1.16 ± 0.14 nmol/mg, mean ± SD) compared to control rats (0.90 ± 0.20 nmol/mg, mean ± SD) (p = 0.0005); however, no change was observed in rats treated at any dose for 28 days (0.961.07 ± 0.070.16 nmol/mg, mean ± SD). MTBE treatment caused a statistically significant 1.5-fold increase in EROD (CYP1A1/2) activity, only in rats dosed with 1500 mg MTBE/kg/day for 15 days (p = 0.0286) (Fig. 2
). No significant difference was noted at any dose group treated for 28 days. A significant increase in PROD (CYP2B1/2) activity (6.5-fold) was measured in rats treated with 1500 mg MTBE/kg/day for 15 (p < 0.0001) and 28 days (p = 0.0427) (Fig. 2
). PNP hydroxylase activity, a substrate marker for CYP2E1, was significantly increased in rats treated with 1500 mg MTBE/kg/day for both 15 (2.3-fold, p = 0.0014) and 28 days (2.0-fold, p < 0.0001) (Fig. 3
). A mild, but significant, increase in PNP hydroxylase activity was also measured in rats treated with 500 mg MTBE/kg/day for 28 days compared to controls (p < 0.0001).
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DISCUSSION |
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In the liver, CYP-catalyzed hydroxylation and subsequent conjugation by glucuronosyltransferases or sulfotransferases is the primary route of testosterone catabolism and elimination (Waxman, 1988). In this study, EROD and PROD, markers for CYP1A1/2 and CYP2B1/2 activity, were increased in male Sprague-Dawley rats dosed with MTBE; however, no changes were observed in CYP2B1 using the formation of 16
-OHT and androstenedione. The increase in CYP1A1/2 and CYP2B1/2 was modest compared to other potent inducers such as 3-methylcholanthrene and phenobarbital which cause a greater than 20-fold induction in their respective CYP enzyme (Parkinson, 1996
). Similarly, PROD activity was increased 50-fold in male Sprague-Dawley rats 18 h following administration of a single intraperitoneal dose of 5 ml MTBE/kg in an unknown amount of corn oil (Brady et al., 1990
). In female B6C3F1 mice exposed to 7814 ppm MTBE for 3 or 21 days or 1800 mg MTBE/kg/day for 3 days (po), CYP1A1/2 and CYP2B1/2 activities were also increased (Moser et al., 1996b
). Other investigators reported no change in total CYP concentration or CYP1A1/2 in male Sprague-Dawley rats exposed to 50, 100, or 300 ppm MTBE 6 h/day for 5 days/week for 2 or 15 weeks (Savolainen et al., 1985
). The highest estimated dose these rats were exposed to, 130 mg/kg/day, indicated that the dose of MTBE was below the lowest dose used in the current study (250 mg/kg) (doses were calculated using a minute ventilation rate of 250 ml/min and a fractional uptake of 0.4). This may explain the lack of CYP induction. In a study investigating the effects of ETBE, a chemical with similar chemical and physical properties to MTBE, CYP2B1/2 was also increased (Turini et al., 1998
). The observed increase in CYP2B1/2 in this study, as measured by PROD activity, was the most sensitive to MTBE administration (6.5-fold). The current study, along with the ETBE study (Turini et al., 1998
), demonstrated an increase in CYP2B1/2 as measured by PROD; however, this CYP was not increased as measured by 16
-OHT or androstenedione formation. The increase in CYP2B1/2 activity measured by the PROD assay may be due to the broad specificity of the PROD enzyme. Although not measured in this study, 16ß-OHT, a marker for CYP2B1/2 activity, has been associated with increased [14C]androgen clearance (Wilson and LeBlanc, 1998
).
Measurement of several testosterone hydroxylase activities indicated that CYP3A1/2 and CYP2A1 activities were increased following MTBE treatment. The mild increase in 6ß-OHT and the absence of an increase in 2ß-OHT, both markers of CYP3A1/2, indicate that this CYP is minimally affected by MTBE administration. No changes were observed in CYP2C11 activity, as measured by 2-OHT, 16
-OHT, and androstenedione. A similar study investigating the effect of ETBE on hepatic CYP enzyme induction showed no changes in CYP2C11 or CYP3A1/2 in male Sprague-Dawley rats following 200 or 400 mg ETBE/kg/day for 4 days or 2 ml ETBE/kg by gavage for 2 days (Turini et al., 1998
). CYP2A1/2 and CYP1A1/2, as measured by 7
-OHT and EROD, were increased in the high-dose rats at 15 but not 28 days. The only explanation for these differences in response with time would be that the rats adapted to the chemical treatment after 15 days.
In the current study, UDPGT activity, a measure of one phase-II conjugation reaction, was significantly increased. In male Wistar rats exposed to 50, 100, or 300 ppm MTBE for 6 h/day and 5 days/week for 2 weeks, a dose-dependent increase in UDPGT activity was measured in hepatic microsomes; however, this response was not observed following a 15-week exposure (Savolainen et al., 1985). Increased UDPGT activity indicates that elimination of testosterone may be enhanced following MTBE administration; moreover, sulfotransferase activity, although not measured, must also be considered when assessing the total clearance of testosterone from serum.
Previous studies with rat and human liver have demonstrated a role of CYP enzymes in MTBE metabolism (Brady et al., 1990; Hong et al., 1997b
; Turini et al., 1998
). MTBE is primarily metabolized in animals to tert-butyl alcohol and formaldehyde by CYP2B1 and to a lesser extent by CYP2E1 (Hong et al., 1999b
; Turini et al., 1998
). In this study, MTBE induced its own metabolism in rats dosed with 1500 mg MTBE/kg/day for 28 days. Brady et al. (1990) suggested that the quantitative increase in CYP enzymes following MTBE administration might affect its oxidation rate. In this study, the induction of MTBE metabolism is consistent with the increase in CYP2E1 and CYP2B1 activity observed in rats dosed with 1500 mg MTBE/kg/day for 15 and 28 days. It is unclear why there were measured differences in the metabolism of MTBE between controls at 15 and 28 days. ETBE also caused an increase in CYP2E1 and CYP2B1 activity in male Sprague-Dawley rats treated with 2 ml ETBE/kg by gavage for 2 days (Turini et al., 1998
). Administration of the metabolite tert-butyl alcohol to male Sprague-Dawley rats did not alter CYP2E1 or CYP2B1 activity following 200 or 400 mg/kg/day (ip) for 4 days (Turini et al., 1998
), suggesting that ETBE, the parent compound, is responsible for the induction of CYP enzymes.
Induction of CYP enzymes responsible for the metabolism of testosterone may play a role in endocrine-related toxicity observed following chronic MTBE administration. Previous two-year cancer bioassays in male rats indicated that MTBE increased the incidence of Leydig cell tumors (Belpoggi et al., 1995; Bird et al., 1997
). The development of these tumors following exposure to nongenotoxic chemicals such as MTBE has been hypothesized to be due to the ability of the chemical to disrupt the hypothalamus-pituitary-testicular (HPT) axis, possibly through the inhibition of testosterone biosynthesis, resulting in a sustained increase in luteinizing hormone (Clegg et al., 1997
). Previous studies demonstrated a modest decrease, not an increase, in serum luteinizing hormone levels in male rats dosed with 1500 mg MTBE/kg for 28 days (Williams et al., 2000
). It is possible that the decreased luteinizing hormone may contribute to the observed decrease in testosterone. Alternatively, a decrease in serum testosterone could result from a variety of different mechanisms that include alterations in the rate of secretion, transport, biotransformation, and elimination of testosterone (Wilson and LeBlanc, 1998
). Chronic stimulation of luteinizing hormone secretion leads to induction of enzymes in the steroidogenic pathway, including some CYP enzymes (O'Shaughnessy and Payne, 1982
; Payne et al., 1980
). For example, results from cisplatin-treated rats indicated that interruption of the HPT axis leads to depletion in circulating androgens, which are necessary for the maintenance of hepatic CYP enzyme expression in male rats (LeBlanc and Waxman, 1988
). Others have postulated that the chemically induced increases in CYP hydroxylase activities are associated with a concomitant increase in testosterone clearance (Clegg et al., 1997
; Wilson and LeBlanc, 1998
). Additionally, endosulfan administration to female mice resulted in increased testosterone hydroxyl metabolite formation with an approximately 3.6-fold increase in the rate of urinary elimination of [14C]androgen and a decrease, albeit not significant, in serum testosterone levels (Wilson and Leblanc, 1998
). This disruption of the HPT axis via enhanced testosterone clearance has also been proposed for felbamate and oxazepam (Cook et al., 1997
), which cause Leydig cell tumors in male rats (Physician's Desk Reference, 1999); there is, however, no direct evidence to support this as a mode of action.
Results from this study indicate that MTBE caused mild increases in testosterone hydroxylase and UDPGT activities, consistent with the observed centrilobular hypertrophy in the livers of treated rats. These changes, along with the decrease in serum testosterone observed in male rats (Williams et al., 2000), suggest that MTBE administration may enhance the elimination of endogenous testosterone. This hypothesis is also supported by the reported induction in CYP enzymes and increased metabolism of estradiol observed in female mice following MTBE administration (Moser et al., 1996b
). It must be considered, however, that the HPT hormonal feedback regulation functions to maintain normal hormone homeostasis and that a mild decrease in testosterone levels via metabolism and excretion would most likely be compensated by the HPT axis regulation. Therefore fluctuations in serum testosterone levels could be minimal, as is the case with MTBE. MTBE induced selected enzymes involved in testosterone metabolism; however, most changes were minimal compared to other known inducers of specific CYP enzymes. We concluded that it is unlikely that the observed degree of induction could be responsible for the decrease in serum testosterone via enhanced clearance.
In addition, several changes were confined to the high doses, which greatly exceed expected human exposure. Caution must be used when interpreting the high-dose responses, since clinical signs of toxicity and decreased body weight gain were observed. Additional studies are necessary to determine whether this induction has a significant effect on the clearance of endogenous testosterone.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at CIIT, 6 Davis Drive, P.O. Box 12137, Research Triangle Park, NC. Fax: (919) 558-1300. E-mail: borghoff{at}ciit.org.
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